|UFDC Home||myUFDC Home | Help|
1 EVOLUTIONAR IL Y CONSERVED CYTOTOXIC T LYMPHOCYTE EPITOPES ON THE HUMAN IMMUNODEFICIENCY VIRUS AND THE FELINE IMMUNODEFICIENCY VIRUS FOR A N HIV 1 VACCINE By MISSA PATRICK SANOU A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2012
2 2012 Missa Patrick Sanou
3 To my father Thomas and my mother Ar le tte. I strive to be like you every day!
4 ACKNOWLEDGMENTS I thank my parents for everything! I thank my sisters Eva and Clotilde, my brother Aristide and my extended family I thank Lionel, Yannick, Aminata and all my friends I will be forever gratefu l to the educational system that I enjoyed in Burkina Faso and benefited from. I give special thanks to all my teachers going back to first grade, especially those who helped nurture my love for the sciences. I thank all the nice people I met along this jo urney from those I only met once to those I had the chance to share more with and know better I thank the University of Florida and the government of the United States of America for giving me the opportunity to accomplish one of my dreams. I thank my fel low graduate students, the members of the ICBR core flow laboratory, my past and c urrent lab members especially Dr Sato, Dr Coleman, and Dr Pu for teaching me the assays. I am grateful to the Department of Microbiology and Cell Science and its staff fo r making our lives easier as graduate students. I thank Dr KT Shanmugam for his time and advice. I am thankful to the people in the Department of Infectious Diseases and Pathology where I did most of my training, especially the front office for their help I also give big thank s to my committee members, Dr John Dame, Dr Joseph Larkin III, Dr Howard Johnson, Dr Ammon Peck, and Dr Janet Yamamoto for their guidance and patience I especially thank my principal investigator, Dr Yamamoto for her time an d advice, the work environment and her dedication to training scientists. I am grateful to Dr Wayne Nicholson and Dr Julie Maupin for having me in their lab for rotation, and also to Dr De Crecy for whom I had been a teaching assistant. I am also gratef ul to Dr Johnson in whose laboratory I had my first immunology rotation and who helped me continue on this path.
5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 7 LIST OF FIGURES ................................ ................................ ................................ .......... 8 LIST OF ABBREVIATIONS ................................ ................................ ............................. 9 ABSTRACT ................................ ................................ ................................ ................... 14 CHAPTER 1 INTR ODUCTION ................................ ................................ ................................ .... 16 HIV 1 Vaccine Trials: Evolving Concepts and Designs ................................ ........... 16 Background ................................ ................................ ................................ ...... 16 Working Towards an HIV Vaccine ................................ ................................ .... 17 Major Phase II and Phase III Clinical Vaccine Trials ................................ ........ 18 Potential Correlates of Protection ................................ ................................ ..... 20 Proposed Methods for Effective Protection against HIV: the T Cell Based Vaccine ................................ ................................ ................................ ......... 23 Multi Epitope Vaccines to In duce T Cell Immunity against HIV ........................ 28 Improvements in Multi Epitope Vaccines ................................ .......................... 32 Selection of HIV Immunogens and Conserved Epitopes ................................ .. 33 Identification of Evolutionary Conserved HIV CTL Epitopes: Use of FIV Proteins ................................ ................................ ................................ ......... 35 Summary ................................ ................................ ................................ ................ 36 2 MATERIALS AND METHODS ................................ ................................ ................ 50 Study Population ................................ ................................ ................................ ..... 50 Ethics Statement ................................ ................................ ................................ ..... 50 HIV 1 and FIV Peptide Pools ................................ ................................ .................. 50 Human ELISpot Analyses ................................ ................................ ....................... 51 CFSE Proliferation Analysis ................................ ................................ .................... 51 Intracellular Cytokine Staining ................................ ................................ ................ 52 Tools ................................ ................................ ................................ ....................... 52 3 CROSS REACTIVITY OF HI V 1 INFECTED SUBJECTS TO EVOLUTIONARY CONSERVED REVERSE TRANSCRIPTASE EPITOPES ................................ ..... 54 Background ................................ ................................ ................................ ............. 54 Inducing Epit opes on HIV 1 and FIV RT ................................ .. 56 Screening for T cell Proliferation Epitopes on HIV 1 and FIV RT ........................... 57
6 tion Responses to Selected HIV and FIV Peptide Pools ................................ ................................ ................................ ...... 58 + T Cell Proliferation Responses ................................ ................................ ............... 59 Characterization of CTL Activities Induced by F3 3 Peptide ................................ ... 59 Determining whether H3 3 and F3 3 are Conserved among Lentiviruses .............. 60 4 CONSERVED VACCINE EPITOPES ON HIV 1 AND FIV P24 RECOGNIZED BY HIV 1 INFECTED SUBJECTS ................................ ................................ .......... 70 Conserved CMI Epitopes Based ................................ ............ 70 Conserved CMI Epitopes Based on T cell Proliferation Responses ....................... 70 CD8 + T cell Proliferation to Fp9 ....... 71 Identifying the Epitope(s) on Fp9 and Fp14 Regions that Induce CMI Responses ................................ ................................ ................................ ........... 72 Characteriz ation of CTL Activities Induced by Fp9 and Fp14 Pools ....................... 73 5 DISCUSSION ................................ ................................ ................................ ......... 78 Identification of CMI Responses on HIV and FIV RT ................................ .............. 78 Evolutionarily Conserved CTL Epitopes on HIV 1 and FIV p24 Proteins ................ 81 Identification and Use of Evolutionary Conserved Epitopes ................................ .... 83 Overall Summary ................................ ................................ ................................ .... 85 LIST OF REFERENCES ................................ ................................ ............................... 87 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 104
7 LIST OF TABLES Table page 1 1 Animal models in HIV 1 research ................................ ................................ ....... 3 8 1 2 Immunogens used in major clinical vaccine trials ................................ ............... 39 1 3 Characteristics of CD4 + and/or CD8 + T cells stimulated by an effective HIV 1 vaccine ................................ ................................ ................................ ............... 40 1 4 Phase I and IIa clinical trials of HIV CTL multi epitope vaccine s ........................ 41 1 5 HIV 1/FIV proteins ................................ ................................ .............................. 43 1 6 CTL epitope predicti on tools ................................ ................................ ............... 44 1 7 Best defined CTL epitopes on HIV integrase ................................ ...................... 45 1 8 HIV 1 integrase CTL epitopes and direct FIV counterparts ................................ 46 2 1 Population characteristics ................................ ................................ ................... 53 3 1 Variation of H3 3/F3 3 aa sequences and immunological responses ................. 62
8 LIST OF FIGURES Figure page 1 1 MHC class I and MHC c lass II molecules. ................................ .......................... 47 1 2 NetCTL 1.2 prediction of HIV SIV, and FIV CTL epitopes. ................................ 48 1 3 Possible location of counterpart epitopes. ................................ .......................... 49 3 1 d FIV RT peptide pools. .................. 63 3 2 Frequency of positive HIV RT (H) and FIV RT (F) peptide pool responses. ....... 65 3 3 Persistence of IFN RT peptide pools. ................................ ................................ ................................ .................. 66 3 4 F3 peptide epitopes recognized by F3 responders. ................................ ............ 67 3 5 Chara cterization of CTL epitopes on F3, H6, F6, and H11 pools. ...................... 68 4 1 HIV and FIV p24 peptide pools. ................. 74 4 2 p24 peptide pools. ................................ ................................ ................................ .................. 75 4 3 Fp and Hp peptide epitope s recognized by pool responders. ............................. 76 4 4 Characterization of CTL epitopes on Fp9, Fp14, and Hp15 pools. ..................... 77
9 LIST OF ABBREVIATION S A alanine aa amino acid Ad5 adenovirus serotype 5 ADCC antibody dependent cell mediated cyto toxicity ANN artificial neural network ANRS agence national de recherche sur le SIDA ART antiretroviral therapy BD Becton, Dickinson and Company bNAbs broadly cross reactive neutralizing antibodies CA California CAEV caprine arthritis encephaliti s virus CFDA SE carboxyfluorescein diacetate succinimidyl ester CFSE carboxyfluorescein succinimidyl ester CMI cell mediated immunity CTL cytotoxic T lymphocyte CTLA 4 cytotoxic T lymphocyte associated antigen 4 D aspartic acid DNA deoxyribonucl eic acid E glutamate EC elite controller EIAV equine infectious anemia virus Env envelope F phenylalanine FACS fluorescence activated cell sorting
10 FIV feline immunodeficiency virus FL Florida G glycine Gag p17 matrix protein Gag p24 core caps id protein Gag p7 nucleocapsid protein GM CSF granulocyte/macrophage colony stimulating factor gp glycoprotein GrzA granzyme A GrzB granzyme B HERV human endogenous retrovirus HESN HIV exposed seronegative individual HIV human immunodeficiency virus HLA human leukocyte antigen HVTN HIV vaccine trials network I isoleucine i.d intradermal i.m intramuscular IAVI i nternational AIDS vaccine initiative ICS intracellular cytokine staining IEDB immune epitope database IFN interferon I N integrase Inc. incorporation K lysine
11 KY Kentucky L leucine LAG 3 lymphocyte activation gene 3 LANL Los Alamos national laboratory LLC limited liability company LT long term LTS long term survivor M methionine MA Massachusetts ME P multi epitope peptide MHC major histocompatibility complex mL milliliter MN Minnesota MVA modified vaccinia Ankara MVV maedi visna virus N asparagine NA not available NAbs neutralizing antibodies NK natural killer nM n anomolar NRTI nucleotide reverse transcriptase inhibitor NY New York OR Oregon PA psoriatic arthritis PBMC peripheral blood mononuclear cell
12 PD 1 programmed cell death 1 Perf perforin PR protease R arginine REF reference RhCMV rhesus cytomegalovirus S IV vectored RNA ribonucleic acid RT reverse transcriptase S serine s.c subcutaneous SA South Africa SIV simian immunodeficiency virus ST short term T threonine T CM central memory T cells TCR T cell receptor repertoire T EM effector memor y CD8 T cells TH T helper Tim 3 T cell immunoglobulin mucin 3 TM trademark TM transmembrane UCSF University of California at San Francisco UF University of Florida UK United Kingdom USA United States of America
13 USF University of South F lorida V valine W tryptophan gamma microgram microliter
14 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philos ophy EVOLUTIONAR IL Y CONSERVED CYTOTOXIC T LYMPHOCYTE EPITOPES ON T HE HUMAN IMMUNODEFICIENCY VIRUS AND THE FELINE IMMUNODEFICIENCY VIRUS FOR A N HIV 1 VACCINE By Missa Patrick Sanou December 2012 Chair: Janet K Yamamoto Major: Microbiology and Cell Scie nce The release of a commercial HIV vaccine for a global population is not yet imminent even after close to three decades of research. Most scientists today believe that both humoral and cell media ted immune (CMI) responses generat ed by immunization will be important in such a vaccine. Efforts to induce those responses take into account the diversity of the virus, as well as the variability of major histocompatibility complex across the globe, especially when it comes to CMI responses. The CMI responses of HIV 1 + long term survivors against the conserved regions of the virus have highligh ted the ir importance in the control of the infection. T hese responses have also indicate d a role of polyfunctional cytotoxic T lymphocytes (CTL s ) and CTL inducing epitopes for an effective HIV 1 vaccine. Furthermore, conserved epitopes were shown to be determinant in an HIV vaccine trial. The most conserved HIV regions are likely to be the best immunogens for inducing protective CTL s as the se epitopes will not easily mutate witho ut a n associated fitness cost to the virus. Cross species recognition has been observed with other viruses and with H IV.
15 The current studies identified conserved regions on HIV by simultaneously comparing the immunological responses of HIV infected individual s to overlapping peptides from HIV and from the feli ne immunodeficiency virus (FIV) The reverse transcriptase enzyme s and viral core capsid proteins of both HIV and FIV were evaluated for their counterpart conserved CTL epitopes. The se studies show ed that the proliferation profile of T cells to HIV and FIV may be more useful for the identification of conserved regions than their induction of interferon g amma secretion. Furthermore, the current approach was able to identify a unique HIV specific epitope, ne ver described before which is highly conserved among lentiviruses and which has the ability to induce CTL cytokines. Therefore, evolutionarily conserved HIV CTL epitopes exist and can be mapped by comparing the immunological responses of HIV infected subje cts to the conserved proteins between HIV and FIV and possibly other lentiviruses.
16 CHAPTER 1 INTRODUCTION HIV 1 Vaccine Trials: Evolving Concepts and Designs Background While developing a successful HIV vaccine would arguably be the best method of eradi cating the AIDS pandemic, designing such a vaccine is proving to be one of the most significant challenges of the 21 st century. Since the discovery of the virus in 1983 , HIV 1 has infected more than 60 million individuals and caused greater than 25 mil lion deaths, most of which have occurred in sub Saharan Africa . Though control of new HIV infections has improved due to major global efforts towards preventive education and access to antiretroviral therapies, new infection rates are still high and th e disease remains a significant burden in countries with high HIV prevalence . Over the past several decades, the primary focus of HIV vaccine research has evolved away from designing a traditional antibody based vaccine towards a more balanced approac h that would activate both humoral and cell mediated immune (CMI) responses in vaccine induced protection [4,5]. New delivery systems, novel immunization regimens, and a wide selection of formulations for immunogens and adjuvants have been developed. It is now quite clear that a careful choice of vaccine immunogens capable of affording protection against many variants of HIV is important. Not only must they protect against existing variants, they must also be able to confront a virus with an extraordinarily high mutation rate, a function that drives a continuum of inc reasing antigenic diversity. The current chapter briefly discusses ongoing research in T cell based HIV vaccines and clinical trials that focused on CMI activation. We also
17 describe a new approa ch to selecting vaccine immunogens based on conserved cytotoxic T lymphocyte (CTL) epitopes. Working Towards an HIV Vaccine While some of the most effective vaccines in use today have been based on attenuating the viral pathogen, concerns regarding the pot ential for reversion to a virulent form of HIV have made the development of an attenuated HIV vaccine unfeasible . Similar concerns related to the safety of inactivated whole viral vaccines have reduced support for a killed HIV vaccine . Challenges t o the development of an effective HIV vaccine based on subunits, rather than whole virus include the enormous diversity of viral sequences and the rapid rate of viral evolution that allows HIV to evade protective immune responses [8,9]. Despite recent succ esses in the identification of broadly cross reactive neutralizing antibodies (bNAbs) against HIV 1, current methods for inducing these antibodies have not been successful . Furthermore, much remains to be learned regarding virus/host interactions and the exact immune correlates of protection; these remain some of the most formidable obstacles to the design of an effective HIV vaccine . Nevertheless, HIV researchers have made substantial progress in understanding the evolution, pathogenesis, an d immunological responses to the virus through preclinical and clinical studies in humans and animals . These advances have been achieved by studying a variety of human populations such as vaccinated volunteers, HIV exposed seronegative individuals (HE SN), and different clinical groups of HIV infected individuals (rapid progressors, elite controllers, and long term survivors or non progressors, see below). Experimental vaccine studies have also been conducted in animals. For example, immune responses to simian immunodeficiency virus (SIV) and
18 recombinant SHIV have been extensively studied in macaques [14 16]. A viral infection that resembles HIV, equine infectious anemia virus (EIAV) has been studied in horses , and similarly, caprine arthritis encep halitis virus (CAEV) has been the subject of extensive research in goats [18,19]. Our group has focused on elucidating immune responses to feline immunodeficiency virus (FIV) in cats [20,21]. More recently, HIV viral vectors have been used to study cell me diated immune responses in rodent models, and the hu SCID mouse has been considered as a potential model for vaccine development [22 25] (Table 1 1). New approaches and technologies utilized in HIV research are advancing science in a multidisciplinary way to face the ongoing scientific challenges of developing both an HIV vaccine and immunotherapeutic strategies. Major Phase II and Phase III Clinical Vaccine Trials VAX004, which was initiated in 1998, was the first phase III human trials to be conducted. T he immunogens for this study were recombinant surface envelope glycoproteins (gp120) from two HIV 1 subtype B viruses (AIDSVAX B/B)  (Table 1 2). The second phase III trial VAX003, initiated in 1999, used HIV 1 subtype B and subtype E glycoproteins (AI DSVAX B/E) as immunogens [27,28]. Both of these vaccines were designed to stimulate humoral immune responses but failed to produce potent neutralizing antibodies (NAbs) against clinical HIV isolates and, consequently, afforded no protective efficacy. Follo wing these two unsuccessful attempts, researchers focused on means of inducing CTL responses to confer protection. These efforts culminated with the STEP vaccine trial (Table 1 2). The vaccine used in this study was designed to induce strong CMI responses . It consisted of an adenovirus serotype 5 (Ad5) vectored HIV 1 gag/pol/nef from three subtype B strains (CAM 1 gag, JRFL pol (RT & IN), IIIB nef) .
19 Initiated in 2004, the phase IIb trial was terminated before completion due to higher HIV infectio n rates among vaccine recipients than among placebo recipients [29,30], possibly explained by the Ad5 sero positivity status and lack of circumcision of the men who became infected [29 31]. This increased risk of HIV infection was shown to fade over time i n a four year follow up study . Outside of the context of vaccination, Ad5 neutralizing antibodies did not increase the risk of HIV infection in a high risk population, even after adjusting for their circumcision status . RV144, which was initiated in 2004, is the most recently completed phase III vaccine trial. The vaccine consisted of a canarypox virus vectored HIV 1 gag/pol/env for priming and the AIDSVAX B/E for boosting. This vaccine was intended to induce both humoral and CMI responses and to cover a wider range of potential challenge strains. The vector expressed both the gag pr gp41 genes from subtype B LAI strain and a gp120 from a circulating recombinant isolate called CRF01_AE. The phase III trial, which involved more than 16,000 subjects, demonstrated only modest overall efficacy (31.2%), and a very minimal efficacy (3.7%) in the high risk group . Detectable antibodies and lymphocyte proliferation responses were observed during the phase II trial, where the majority of vaccine recipien ts also demonstrated antibody dependent cell mediated cytotoxicity (ADCC) responses [35,36]. Following phase III, IgG antibodies binding to the V1/V2 variable loops of Env were found to correlate with protection, while serum IgA to Env correlated with incr eased risk of infection without causing an enhancement of infection rates [37,38]. In summary, during the past three decades of HIV vaccine research, only three candidate vaccines have completed phase III clinical trials , reflecting the need for
20 addi tional research and development of innovative approaches. Furthermore, in these phase III trials, only the HIV 1 subtype B (three strains) and a recombined HIV 1 subtype A/E (gp120 from CRF01_AE) were used as vaccine immunogens. Although these strains and subtypes reflect the circulating forms of HIV that are more prevalent in the geographical location of the trials, they may not contain optimal immunogens needed for a vaccine effective against a wider range of HIV isolates. The relative success of the RV14 4 trial suggests that an HIV vaccine may be possible using current technologies. In the next few sections, we address aspects of HIV vaccine development and propose a path forward that may improve on this modest su cces s. Potential Correlates of Protection An immune correlate of protection can be defined as an immune response that is normally associated with protection from infection or disease and for which a measurable threshold can be defined . In the case of HIV/AIDS, the study of HIV exposed serone gative individuals (HESN) and HIV positive (HIV ) elite controllers (ECs) has provided significant insights into the immune correlates of protection from infection and disease. Furthermore, studies of the RV144 trial have made contributions by defining mor e precise vaccine induced immune correlates of protection in humans. When fully identified these correlates should provide a turning point for advancing HIV vaccine development . HIV exposed seronegative is a term that refers to cohorts of commercial s ex workers, hemophiliacs, discordant couples, intravenous drug users, and mother to child cases, who, despite having high risks or documented exposures to HIV, have remained uninfected . While a range of factors can potentially explain how HESN remain uninfected, a range of conditions including genetic mutations, T cell specific HIV
21 responses, CD8 + cell antiviral activities, components of innate immunity, or intracellular intrinsic factors have been considered, but conclusions regarding the causes of th ese conditions are not evident. Efforts are currently underway to standardize the above studies and assays and explore other avenues [42,43]. Elite controllers have been a major focus of many studies. They are HIV individuals who are able to maintain undetectable viral loads (<75 RNA copies/mL) for many years without the use of antiretroviral therapy . These individuals are relatively rare, representing less than 1% of all HIV infections . The majority of ECs exhibit even slower rates of CD4 T cell decrease and slower disease progression than regular long term non progressors [46 48]. CD8 T cells play a central role in controlling HIV replication in ECs [49 51]. Selected genetic factors have also been ass ociated with an elite control of the viral infection, including the CCR5 co receptor 32 base pair deletion or the killer cell immunoglobulin like receptors of NK cells [48,52]. HLA B57 and B27 are also over represented in EC populations and have been corr elated with a lower risk of disease, as opposed to certain HLA B35 alleles which are associated with a higher risk for disease progression [52,53]. Recent results of the International Controller Study, in addition to confirming previous findings, showed th at the major genetic determinants associated with control of HIV infection are located in the peptide binding groove of the MHC class I molecule (primarily HLA B), and also implicated HLA C and other polymorphisms linked to NK cells . Though these gene tic factors can explain about a fifth of the variability in virus control, the question remains to be answered on whether the induction of the same cellular responses can be elicited in individuals who carry different HLA alleles . Moreover, HLA B57 an d B27 are associated with rapid
22 disease progre ssion in some individuals . CTL and NK cells can kill virus infected cells by releasing cytotoxins contained in their granules: granulysins and perforin make openings on the cell membrane allowing granzyme s which are serine proteases to enter and induce cell death by apoptosis. CTLs are able to detect virus infected cells through their abilit y to recognize a complex of viral antigen presented by an MHC molecule (Figure 1 1). CMI responses have been associa ted with the initial control of HIV infection and reflected a non cytotoxic response against the virus . The important role of the CD8 CTLs in the control of HIV disease has subsequently been reported [58 61]. In addition, CD4 CTLs play an important role early in the control of other viral infections [62,63]; these have also been shown to be relevant in the case of acute HIV infections . Furthermore, a study of rabies vaccination has demonstrated that NK cells can serve as effectors in acquired or adaptive immunity as a result of CD4 T cell activation . Similarly, NK cells have been described in other viral infections to possess key adaptive immune features such as memory response and antigen specific proliferation [66 68]. Consequently their involvement in vaccine design is beco ming better appreciated. With respect to HIV, NK cells have also been associated with the prevention of mother to child and heterosexual HIV transmission [69,70]. Some studies suggest that they may play a role in the control of HIV replication by respondin g to peptides [69,71], and may be influenced by the peptide/HLA B complex via the KIR3DL1 and KIR3DS1 molecules . The role of antiviral antibodies in HIV infection has been demonstrated by passive transfer studies with NAbs, which showed that anti HIV NAbs prevented SHIV
23 infection in macaques  and by anti HIV NAbs that delayed virus rebound after interruption of antiretroviral therapy (ART) in humans . However, ECs showed no significant difference in NAbs when compared to patients on ART . In addition only low levels of bNAbs have been observed in the majority of ECs, a population that is heterogeneous in terms of immunological characteristics [75,76]. Nevertheless, while ECs can have strong CD8 cell mediated anti HIV responses, their antiviral antibody responses may still play a role in the control of HIV replication [77,78]. Proposed Methods for Effective Protection against HIV : t he T Cell Based Vaccine A successful HIV vaccine that is able to control the AIDS pandemic could have a infection that is later cleared; or a long term control of the virus without disease manifestations and without further HIV transmi ssion from the vaccinated individuals. The more conservative and accepted view is that both humoral and CMI responses will be important in such a vaccine. Based on the lessons learned from successful vaccines against other organisms, one correlate of prote ction has been a defined antibody titer directed against one of the key antigens of those organisms . This correlate is usually derived from the assessment of immune responses in recovered patients who are immune to subsequent infections and serves as a benchmark titer for the development of an effective vaccine. With those conventional vaccines and immune individuals, the induction of specific antibodies is the probable cause of protection against future infections . Although CD4 T cells participate in the B cell response and memory, T cells in general are thought to play a more dominant role in the control of established infections rather than in prophylaxis .
24 Induction of antibody response may be critical to developing an ef fective vaccine, but it has been difficult to identify the proper immunogen. Due to the mounting difficulties associated with making a conventional antibody based HIV vaccine , some researchers have returned to exploring T cell based approaches. Design ing T cell mediated vaccines requires careful planning so as to generate immune responses in the context of most human leukocyte antigens (HLA). HLA molecules differ from one individual to another and their prevalence also varies in different populations, a factor that has been a cause for concern about T cell mediated vaccines . However, HLA class I and class II alleles can be grouped into supertypes which are clusters of HLA molecules sharing overlapping peptide binding specificity [82,83]; vaccines c ontaining epitopes that can be presented by HLA supertypes have a better chance of global success. Three HLA class I supertypes (HLA A2, A3, and B7) have the potential to cover 80 90% of the major ethnicities . Studies have shown that not all CTL respo nses are equally effective. For example, inclusion of epitopes presented by some B7 supertype alleles targeting the same epitopes may exert differential pressure on the virus . Nonetheless, a T cell based vaccine using the supertype concept may be able to induce similar qualitative T cell responses against the virus among individuals with diverse HLA alleles. Most HIV infections occur via sexual transmission at the mucosa , which is a replication site and a reservoir for the virus [86,87]. Therefor e, if CMI is important, an effective vaccine should induce protective responses at the mucosal surface where the Some researchers have reported that CD8 CTLs from the blood and mucosa mirror each other  wi th some of them originating
25 from the same clones or sharing the same HIV specific epitopes and MHC restrictions . In chronically infected individuals, antigens recognized by mucosal T cells are also recognized by peripheral blood mononuclear cells (PBM C) with a few exceptions [91,92]. As a result, the study of these individuals may help define some important correlates of protection that are relevant against HIV at the mucosa. Findings from non human primate studies have shown that the picture of the s ystemic immunity obtained from the study of PBMCs may only be a snapshot of the immunity present at the mucosa [93,94]. Notably, in a study recapitulating human HESN where macaques were intentionally exposed to low doses of SIV rectally, potent virus speci lamina propria mononuclear cells in the absence of detectable virus and virus specific humoral and T cell responses in the blood. Importantly, when these responses were present, they were universally associated with protection from mucosal challenge with much higher doses of virus . One caveat still remains at which site immune correlates of virological control can be identified. In a recent macaque therapeutic trial assessi ng the effect of a particle mediated DNA vaccine administered during antiretroviral therapy, it was shown that the breadth of epitope recognition observed in the mucosa, but not the blood, correlated with control of virus rebound when therapy was stopped [ 96]. Such findings emphasize the need for parallel vaccine trials in macaques and humans to better understand the role of mucosal T cell responses in protection and help define surrogate correlates of protection in the blood of humans. Based on the studies of HIV individuals, a protective T cell based HIV vaccine should have a set of defined characteristics  (Table 1 3). The vaccine should
26 stimulate both CD4 + and/or CD8 + T cells [97,98], which should have a large T cell receptor repertoire (TCR) diversity [99 ], an ability to secrete multiple cytokines , proliferate , destroy HIV infected cells or at least suppress viral replication ex vivo [102,103]. These cells should express markers for central or effector memory functions [104,105], and should not express markers of T cell exhaustion . They should be specific for several Gag epitopes [107,108] restricted by HLAs associated with viral control  and conserved across subtypes , particularly since mutations in Gag would impair the fitness of the virus . This extensive list of attributes are only associated with cases of better disease outcome and highlights the difficulties of making a vaccine based on T cell immunity. Lessons learned from the major trials can also help design better T cell vaccines. The possible explanations for the lack of efficacy in the STEP study are important factors to consider in the context of the anti HIV immunity generated. Pre existing Ad5 specific cellular responses conserved among many adenoviruses were shown to mitigate T cell responses against the HIV 1 insert . Individuals with lower Ad5 neutralizing antibodies before vaccination generally had higher T cell responses to one or more HIV 1 proteins . Furthermore, the vaccine induced a lower freq uency of the T cell responses against the conserved HIV epitopes than against the variable epitopes . The few favorable notes from the STEP trial are also factors to consider. Lower HIV 1 RNA levels were observed in vaccine recipients carrying HLA all eles associated with control of the virus than for the matched placebo recipients . The vaccine used in the STEP trial (mainly a population of men who have sex with men) was simultaneously tested in a heterosexual cohort in South Africa. In this study the
27 increased risk of HIV 1 infection was not observed, even at higher titers of pre existing Ad5 neutralizing antibodies in men. In contrast, favorable CD4 T cell counts were observed at 2 and 3 months after infection, and hints of lower viral set points, were observed in women . These findings indicate that a careful choice of vaccine vector, with focus on conserved HIV 1 epitopes, and efforts to incr ease the breath and magnitude of the responses against HIV are likely to improve future T cell vaccines . The Env antigen in an Ad26/MVA vector vaccination has been shown to be essential for protection against a heterologous neutralization resistant S IV challenge in primates where protection correlated with Env binding antibodies . A rhesus cytomegalovirus SIV vectored (RhCMV) vaccine without Env was able to control a pathogenic SIV below detectable levels for more than a year. This study also sho ws that a replicative vector like RhCMV can induce SIV specific effector memory CD8 T cells (T EM ) important for early protection, while a non replicative vector like the Ad5 induces central memory T cells (T CM ), which is useful in the control of viremia [ 120]. In the human RV144 vaccine trial where IgG antibodies to Env correlated with protection, the vaccine induced stronger humoral than cellular immune responses, along with a predominance of CD4 T cells [34 36] including polyfunctional CD4 CTL against a V2 Env region . Therefore, a vaccine design optimized for also inducing CD8 T cell responses in addition to the CD4 T cell and humoral responses is likely to improve on the current success.
28 Multi Epitope Vaccines to Induce T Cell Immunity against HIV A multi epitope vaccine can be defined as a vaccine construct containing a string of individual epitopes, designed to elicit targeted immune responses to the selected epitopes, which can be the most conserved or the most recognized by common HLAs [41,1 22,123]. Computational methods for minimizing the generation of junctional immunogenicity in between epitopes have been used to construct epitope strings . Multi epitope vaccines have been shown to be protective in animal models and in mice against le thal challenge doses of vaccinia virus [124 126]. There are relatively few multi epitope CTL vaccines that have undergone phase I and II clinical trials against HIV. These vaccines were generally safe, but poorly immunogenic (Table 1 4), most likely due to the vaccine vehicle and the limited number of epitopes contained in the vaccine. The traditional interferon vaccine trials because of its relative efficiency, and has been the main method of assessing HIV specific T ce ll immunogenicity in the majority of the trials described in Table 1 4. A multi epitope vaccine consisting of a concensus subtype A Gag p24/p17 linked to a stringed gene of CTL epitopes (termed HIVA) has been evaluated in a number of different trials from the International AIDS Vaccine Initiative (IAVI) including prime boost designs, as opposed to the HIV Vaccine Trials Network (HVTN) and the Agence National de Recherche sur le SIDA (ANRS) trials described in Table 1 4 [127,128]. The initial phase I studie s of the HIVA vaccine were conducted in the UK (IAVI 001, 003, and 005) [129,130], in parallel with sister studies in Kenya (IAVI 002, 004, and 008) and in Uganda (IAVI009, phase I/IIa) . Contrasting results were associated with these trials. Even tho
29 vaccine induced responses in a higher percentage of vaccinees in the UK (78%, 88%, and 89%) than in Kenya (15%, 33%, and 10%) or Uganda (13%). Subsequent phase I/IIa studies of IAVI 006 and 010  that were conducted with a larger number of volunteers also showed poor vaccine immunogenicity, two weeks after the last MVA vaccination. These results could have been due to low antigen doses or to the timing for testing the immunogenicity, as the responses were later observed to peak one week after the MVA vaccination, and to diminish by the second week. In the IAVI 016 trial, four of eight (50%) DNA MVA vaccine recipients showed ELISpot in which PBMCs were stimulated with the target antigens and cultured for 11 to 13 days prior to being tested for immune responses to epitopes in the ELISpot assay. This procedure improved detection of immunogenicity. A similar approach was recently used in vaccine induced protection . Subsequently, in the IAVI 016 trial, eight of eight (100%) volunteers who received the prime boost vaccination and four of eight (50%) wh o received two doses of the MVA vaccine showed CD4 T responses. In addition five of the 12 (42%) responders also had CD8 T cell specific Another trial (HVTN 048) consisted of four immuniza tions of a DNA vaccine (expressing a 277 aa antigen) containing 21 CTL epitopes. Immune responses to the epitopes were monitored up to 18 months after the last immunization. Only 1 of 8 (13%) recipients of the highest dose of vaccine (4 mg) showed a positi ELISpot
30 response two weeks after the last immunization. Notably, only three CD8 CTL responses were transiently detected using the chromium release assay . In the HVTN 056 trial, the immunogen was a multi epitope peptide (MEP) vaccine consisti ng of four peptides (27 47 aa) supplemented with an adjuvant in the presence or absence of granulocyte/macrophage colony stimulating factor (GM CSF). Initially intended as a priming agent for a prime boost vaccine regimen, the vaccine was only weakly immun second or third vaccination. The use of GM CSF in the formulation did not improve the immunogenicity of the vaccine . The ANRS VAC18 was a phase II trial consisting of four intramuscular injections of a lipopeptide vaccine. This vaccine contained an equal weight mixture of 5 HIV 1 peptides (19 32 aa) linked to a palmitic acid for easy uptake by antigen presenting cells. A cultured Regardless of the vaccine repeatedly detected in the majority of vaccine recipients for 12 months after the last immunization. Proliferative responses were also detected to th e CD4 T cell epitopes . This lipopeptide vaccine was also used in a post infection immunization study, where it induced new CD4 T cell proliferation and CD8 T 1 infected subjects . The vaccines described above and in Table 1 4 used only a modest number of selected CTL epitopes (21 77). In general, these vaccines were unable to induce the desired breadth of CD8 CTL responses. This may be related to the HLA restriction of the study subjects, even though the multi epito pe vaccine HVTN 048 and HVTN 056
31 trials required immunized subjects to carry at least one of the HLA alleles targeted by the vaccine. In the IAVI 009 trial, the vaccine was expected to induce at least two to three CTL epitope responses per individual. An i mportant concern related to these vaccines is whether the numbers of epitopes used will suffice to induce an effective protection against a wide range of global HIV isolates. The impact of immunizing study subjects against variant viral isolates cannot be tested until a sufficient level of immunogenicity is obtained for the vaccine epitopes in the target populations. A different clinical trial (HVTN 064) used a multi epitope vaccine consisting of 18 T helper epitopes formulated in aluminum hydroxide. This T helper vaccine was immunogenic in 32 of 47 individuals (68%) by intracellular cytokine staining, and the CD4 T cell responses observed were polyfunctional. However when administered simultaneously with the same DNA vaccine used in the HVTN 048 trial, this vaccine was unable to improve the immunogenicity towards the CTL epitopes. This may be related to the initi al inability of the DNA vaccine alone to induce sufficient CTL responses . The HIVA prime boost vaccination approach has also been used as a post infection immunization strategy in phase I trials for HIV 1 infected subjects. Despite initial concerns that immunization might boost viral titers, the vaccine was safe and effectively induced an expansion of CD4 and CD8 T cells specific for vaccine epitopes without viral load rebound [141,142]. Another study utilized the ELISpot assay to T cells following stimulation with either the MVA.HIVA vector or HIV 1 peptides used in the construct. The PBMCs obtained from 1 peptides . An
32 additional two clinical trials have recently te sted the safety and immunogenicity of HIVA DNA MVA vaccination in 48 healthy Gambian infants born to HIV 1 uninfected mothers (NCT00982579 trial) and in 72 healthy Kenyan infants born to HIV 1 infected mothers (NCT00981695 trial). These results are current ly pending. Improvements in Multi Epitope Vaccines Priming with a DNA vaccine and boosting with a viral vector vaccine has previously been shown to be an effective way of inducing immunogenicity. Nevertheless, despite the rational design and the use of a s tring of CTL epitopes, the HIVA prime boost vaccination failed to induce strong CTL responses in the IAVI trials (Table 1 4). The string of CTL epitopes optimized for processing in a DNA vaccine (HVTN 048) as well as a peptide vaccine supplemented with adj uvant and GM CSF (HVTN 056) also failed to induce the desired responses in the vaccinees. These results lead to concerns about the multi epitope approach for designing an effective HIV vaccine. However, a number of methods for improving vaccine immunogenic ity and stimulating CD8 T cells have yet to be investigated in clinical trials. They include, and are not limited to the following: the enhancement of epitope by increasing the affinity or cross reactivity of the epitopes for MHC and/or TCR ; the use of multiple cytokine combinations (GM CSF + IL 12; CD86 + GM CSF + IL 12; IL 15) [145 147]; the use of toll like receptor (TLR) modulators such as CpG for DNA vaccines ; the use of alternate vaccine types and routes of administration [35,149 151] inc luding autologous dendritic cells as a vaccine delivery system . Several approaches might be used in combination, leading to improved immunogenicity of multi epitope vaccine candidates. Multi epitope vaccines can be designed to include HLA supertypes for populations with a diverse genetic background, and promiscuous epitopes can help limit
33 the number of final epitopes without decreasing the genetic coverage. Multi epitope vaccines can include both dominant and subdominant epitopes, while avoiding anti genic elements that may not be favorable for protection. Even moderate successes with this type of vaccine against HIV might lead to improvements in future multi epitope vaccine designs, not only against HIV but also against other pathogens. Selection of H IV Immunogens and Conserved Epitopes Careful design of vaccine immunogens for protection against a wide number of HIV variants will be required to deal with the large antigenic diversity. Conserved viral antigens, subtype matched antigens, consensus antige ns, variants of single antigens and multiple antigens have all been used alone or in combination [152,153]. Table 1 4 shows a few examples for each of the strategies. Cross clade CTL responses have been shown to preferentially target the conserved regions over the more variable ones , and these responses have been associated with better HIV disease outcomes or no disease manifestation [107,155,156]. The most conserved regions of HIV, especially those conserved across subtypes  or among lentiviruse s , may be the best targets of the immune system for inducing vaccine protection. Some of these regions may be protective and are less likely to mutate because they hold a functional or structural importance to the virus species (possibly to the genus) ; a mutation would induce impairment to viral fitness [157,158]. This possibility makes the identification of conserved epitopes an important aspect of immunogen selection in vaccine design. One means of including these conserved regions is to construct po lyvalent mosaic proteins as vaccine immunogens; thus far, preclinical evaluations of the mosaic vaccine have demonstrated great potential for broad T cell responses, across subtypes [153,159,160].
34 A method of selecting highly conserved regions is to ident ify those with the lowest entropy, which is the lowest variability at each aa position. Based on this concept, the most conserved HIV proteins have been shown to be (in order of lowest variability): integrase (IN), core capsid (Gag p24), reverse transcript ase (RT), and protease (PR) (Table 1 5) . They were followed by Vpr, Vif, matrix (Gag p17), Nef, Rev, and the surface envelope (SU Env). Tat and Vpu have the highest variability (Table 1 5). This observation suggests that the selection of conserved va ccine epitopes should be done first from IN, Gag p24, RT, and PR. While Jenner may not have considered functional conservation when developing his smallpox vaccine, he can be considered to have been the first developer of a vaccine that was based on conse rved features between two different viral species . In a similar fashion, comparisons with other lentiviruses could help identify highly conserved epitopes that are required for viral function and survival. FIV is a lentivirus that is only distantly r elated to HIV 1, but may still be relevant to the evolutionary conserved approach of vaccine development because of the shared similarities between the HIV and FIV viruses in terms of aa sequence, structure, and pathogenesis . A comparison of the aa co mposition of proteins between HIV 1 and FIV demonstrates the following percentages of identity/homology: RT, 47/72; IN, 37/65; Gag p24, 32/63; nucleocapsid (Gag p7), 30/54; PR, 24/48; Gag p17, 20/50; SU Env, 19/43; transmembrane envelope (TM Env) 18/42 [21 ] (Table 1 5). The three most conserved proteins are also those that have the lowest entropy calculation, as shown in Table 1 5 . Hence, the IN, RT, and Gag p24 proteins appear to be excellent targets
35 for identifying evolutionary conserved regions tha t may also contain conserved T cell epitopes. Identification of Evolutionary Conserved HIV CTL Epitopes: Use of FIV Proteins Immunoninformatics has become an integral part in the design of new vaccines with great promise of rapid and effective vaccine disc overy [163,164,165]. A number of tools and databases are now available online including HLA class I and II binding predictions [166,167], and a number of tools that are useful for the prediction of CTL epitopes (Table 1 6). In one study performed by our g roup, NetCTL 1.2 was used to identify CTL epitopes on the integrase sequences of HIV, SIV and FIV (Figure 1 2 A). For the twelve HLA supertypes shown in Figure 1 2 a large number of CTL epitopes were predicted on each integrase sequence regardless of the v irus: HIV with 78 epitopes, SIV with 74 epitopes, and FIV with 85 epitopes. Some of these were conserved between HIV and SIV (34 epitopes), as well as between HIV and FIV (25 epitopes) (Figure 1 2 B). A smaller number (17 epitopes) was conserved among all t hree viruses, reducing the target epitopes to the expected most evolutionary conserved. Thirteen HIV CTL epitopes termed best defined CTL epitopes have been identified empirically on HIV integrase by different laboratories and compiled on the Los Alamos Na tional Laboratory (LANL) website (Table 1 7). In this regard, based on observations using SIV and FIV, an evolutionary conserved HIV CTL epitope can be defined as a CTL epitope with a direct or indirect SIV and/or FIV CTL counterpart (Figure 1 3 ). Using th e direct counterpart approach (Figure 1 3 arrow a), three of these epitopes are predicted to be CTL epitopes conserved between HIV, SIV, and FIV and one was shown to be an indirect FIV counterpart (Table 1 8). They share the same HLA binding and CTL super type predictions [169,171]. As illustrated in Figure 1 3 an
36 evolutionary conserved epitope may be located on an HIV protein different from that of FIV (Figure 1 3 arrow c). An indirect counterpart to an HIV epitope (bolded in Table 1 8), located upstream on FIV integrase, has higher aa identity and homology than the direct counterpart (Figure 1 3 arrow b). This indirect FIV counterpart has the same binding alleles and predicted CTL supertype as the HIV epitope. The predicted results of SIV sequences can be explained by the high aa identity between HIV and SIV as SIV is more closely related to HIV than FIV. However, despite the relatively lower aa identity between HIV and FIV, FIV counterpart epitopes still appear to be potentially effective HIV antigens ( see Table 1 8), most likely due to the slightly higher aa homology observed between the two viruses. This finding indicates the strong potential that both SIV and FIV epitopes could induce CTL responses in human PBMCs and suggest that this type of comparis on may lead to the identification of evolutionarily conserved CTL epitopes on HIV. Therefore, conserved SIV and FIV IN peptides can be used as immunogens in vitro to compare and identify conserved immune responses generated by the PBMCs of HIV individuals Summary An effective prophylactic HIV vaccine should probably include both humoral and CMI responses, since antibodies (bNabs, ADCC antibodies) and CTLs are likely to play critical roles in the control of the virus. HIV epitopes that are highly conserved are believed to be relevant to viral fitness and thus important in the design of a global HIV vaccine. The identification of evolutionary conserved linear epitopes is feasible for both B and T cells with currently available bioinformatic tools, but most b NAbs do not usually target linear epitopes. Therefore this approach is more useful for identifying T cell
37 epitopes. Testing for immune responses in HIV individuals and vaccinated volunteers from clinical trials can help determine the relevance of these ep itopes in protection against the virus. Conserved CTL epitopes could be used to supplement HIV protein vaccines or used as part of a prime boost combination, which has so far provided the best efficacy in a phase III trial. An effective prophylactic T cell vaccine containing highly conserved epitopes may inform therapeutic strategies and help the millions of HIV infected individuals to effectively control viral replication with or without ART.
38 Table 1 1. Animal models in HIV 1 research Animal models Virus Major cell types infected Major clinical disorders REF b Macaques SIV/SHIV Macrophages, CD4 T cells Immune deficiency, AIDS like [14 16] Cats FIV Macrophages, CD4 T cells, CD8 T cells Immune deficiency, AIDS like [20,21] Horses EIAV Macrophages Aut oimmune hemolytic anemia  Goats CAEV Macrophages Arthritis Encephalopathy [18,19] Rodent models (e.g., hNOG) a HIV 1 (vectors) Macrophage like cells, CD4 T cells CD4 T cell loss [22 25] a humanized NOD/SCID/IL2Rnull (hNOG): severely immunodefici ent mice that can easily engraft human cells. b Reference number (REF).
39 Table 1 2. Immunogens used in major clinical vaccine trials Vaccine Trial Phase (Vaccine name) Trial Location HIV 1 immunogens (Vaccine type) Trial Outcome REF a VaxGen 004 Phase III (AIDSVAX B/B) North America Subtype B gp120s (subunit proteins) No efficacy  VaxGen 003 Phase III (AIDSVAX B/E) Thailand Subtypes B & E gp120 (subunit proteins) No efficacy [26, 28] Step HVTN 502 Phase IIb (MRKAd5 HIV 1) Americas Subtype B Ad5 gag/ pol/nef (Ad5 vector ) No efficacy (enhancement of infection) [29,30] RV144 Phase III (ALVAC HIV 1 AIDSVAX B/E) Thailand Prime subtype B and A/E ALVAC HIV gag pr gp41 gp120 (canarypox vector) Boost Subtypes B & E gp120 (subunit proteins) Some efficacy [General population (31.2%) [ High risk groups (3.7%)]  a Reference (REF).
40 Table 1 3. Characteristics of CD4 + and/or CD8 + T cells stimulated by an effective HIV 1 vaccine Characteristics REF b Have a large T cell receptor repertoire diversity  E xpress markers of central and effector memory functions [102,103] Be able to destroy HIV 1 infected cells or suppress HIV 1 replication ex vivo [100,101] Be able to proliferate upon HIV antigenic stimulation  Be polyfunctional in cytokine production upon HIV antigenic stimulation  Be specific for several Gag epitopes that are restricted by HLAs associated with viral control and conserved across many subtypes [106 109] Not express markers of T cell exhaustion (e.g., PD 1, LAG 3, Tim 3, and CTLA 4) a [104,110 112] Not be specific for the envelope glycoprotein (Env)  a Programmed cell death 1 (PD 1); lymphocyte activation gene 3 (LAG 3); cytotoxic T lymphocyte associated antigen 4 (CTLA 4); T cell immunoglobulin mucin 3 (Tim 3). b Reference (REF).
41 Table 1 4. Phase I and IIa clinical trials of HIV CTL multi epitope vaccine s Trial a Site (# subjects enrolled in the study) b Vaccine type (Regimen) c Dose /route DNA (mg), MVA (p.f.u.) d HIV Antigens (# of CTL epitopes) e HIV subtypes i nvolved f HLA super type of CTL epitopes Epitope selection method (%) g Respon ders REF h IAVI 001 UK (18) DNA (d: 0,21) 0.1 or .05 mg / i.m. p24/p17 gene [contains TH epitopes] + 24 CTL epitopes [p24(6),pol(6), nef(8),Env (4)] A* [A ,B,C,D, E,F,G,H] j A2 ,A3, A24, B7, B8, B27, B44 Most common HIV subtype in Kenya Conserved epitopes 78% [ 121] IAVI 002 Kenya (18) DNA (d: 0,21) 0, 0.1 or 0.5 mg / i.m. 15%  IAVI 003 UK (8) MVA (d: 0,21) 5x10 7 p.f.u. / i.d. 78%  IAVI 004 Kenya (18) MVA ( mo: 0,1) (mo:0) 0 or 5x10 7 p.f.u. / i.d. 25%  IAVI 011 Switzerland/ UK/ SA/(81) MVA (mo: 0,2) 0, 5x10 6 5x10 7 or 2.5x10 8 p.f.u. / i.d., i.m. or s.c. 6%  IAVI 005 UK (9) p DNA (d: 0,21) i b MVA 0.1 or 0.5 mg / i.m. 5x10 7 p.f.u. / i.d. 89%  IAVI 006 UK (119) p DNA (mo: 0) b MVA (mo:2, 3 or 5,6) 0, 0.5 or 2 mg / i.m. 0 or 5x10 7 p.f.u. / i.d. 12%  IAVI 008 Kenya (10) p DNA (d: 0,21) b MVA (mo: 9, 10) 0.5 or 1 mg / i.m. 5x10 7 p.f.u. / i .d. 10%  IAVI 009 Uganda (50) p DNA(mo: 0,1 or 0) b MVA (mo: 5,8) 0 or 0.5 mg 1x or 2x/ i.m. 0 or 5x10 7 p.f.u. / i.d. 15%  IAVI 010 Kenya/UK (114) p DNA (mo: 0,1) b MVA (mo: 5,8) 0.5 mg / i.m. 0, 5x10 6 5x10 7 or 2.5x10 8 p.f.u. / i.d. 3%  IAVI 016 UK (24) p DNA (mo: 0, 1) b MVA (mo: 2 or 0,1) 0 or 4 mg / i.m. 0 or 2.5x10 8 p.f.u. / i.d. 50% 
42 Table 1 4. Continued Trial a Site (# subjects enrolled in the study) b Vaccine type (Regimen) c Dose /route DNA (mg), MVA (p.f. u.) d HIV Antigens (# of CTL epitopes) e HIV subtypes involved f HLA super type of CTL epitopes Epitope selection method (%) g Respon ders REF h HVTN 048 USA, Bostwana (36) DNA (mo: 0,1,3,6) 0.5 mg 4x / i.m. 21 CTL epitopes [Gag(4), Pol(8), Vpr(1), Nef(2), Rev(1), Env(5)] + TH epitope (1 pan DR) A, B, C, D, AE, AG A2, A3, B7 Conserved Epitopes HLA cover age 0%  2 mg 4x / i.m. 0% 4 mg 4x / i.m. 13% HVTN 056 USA (40) MEP [peptides + adjuvant] (mo: 0,1,3) adjuvant 3x / i.m. 4 peptides (55 CTL epitopes): Env TH/Gag CTL(5) Gag TH/GagCTL(19) Env TH/Nef CTL( 15) Env TH /Gag CTL(16) B* A1, A2, A3, A24, B7, B8, B27, B58, B62 Epitope clustering on LANL 13%  USA (40) MEP [peptides + adjuvant + GM CSF ] (mo: 0,1,3) adjuvant + 50 ug GM CSF 3x / i.m. 3% ANRS VAC18 France (99) Lipopeptides (mo: 0, 1, 3, 6) 5 lipopeptides (77 CTL epitopes, containing 7 TH epitopes): [Gag1(9), Gag2(21), Nef1(16), Nef2(21), Pol (10)] A1, A2, A3, A24, B7, B8, B27, B58, B62 Conserved regions 71% k 60% k 70% k  a All trials are phase I clinical trials except for the bolded trial numbers which are phase IIa (with subjects not at risks of HIV infection); International AIDS Vaccine Initiative (IAVI); HIV Vaccine Trials Network (HVTN); Agence National de Reche rche sur le SIDA (ANRS). b United Kingdom (UK); South Africa (SA); United States of America (USA). c Prime (p); boost (b) ; day (d); month (mo); modified vaccinia Ankara (MVA); multi epitope peptide ( MEP ); granulocyte macrophage colony stimulating factor (G M CSF). iNine of the 18 volunteers from IAVI 001 who were primed with HIVA DNA agreed to receive a boost 9 14 months later. d Intramuscular immunization (i.m.); intradermal immunization (i.d.); subcutaneous immunization (s.c.). e MHC class I molecules ca n accommodate CTL epito pes of 8 to 11 aa in length [137 ]. The p24/p17 represents 73% of the Gag and contains both CTL and T helper epitopes. The pan DR T helper epitope is a 13 mer that binds to all common HLA DR alleles. Each of the four peptides in the M EP vaccine is made up of both TH and CTL epitopes; T helper (TH). f The HIV subtypes used in the vaccine. *Consensus sequence. j The CTL epitopes are present in 50 90% of HIV isolates from the different subtypes. g Percentage of vaccinees with detected IFN after the end of the immunization schedule for the IAVI studies; after the last immunization for HVTN 064; and after the 2nd or 3rd vaccination (single time point) for HVTN 056. k Cultured ELISpot assay results. h Reference (REF).
43 Table 1 5. HIV 1/FIV proteins IN Gag P24 RT PR Gag P17 SU Env REF c Approximate average entropy scores a 0.16 0.18 0.21 0.23 0.45 0.6  HIV/FIV protein % aa identit y/homology b 37/65 32/63 47/72 24/48 20/50 19/43  a The average Shannon entropy score is the average value of variability of a given protein at each aa position, calculated by using many aligned sequences. The approximate values shown are derived from the figure of HIV 1 (group M) protein variability from Yusim et al , where the proteins are presented from lowest to highest variability. Lower scores represent lower variability and therefore higher aa conservation. b The percentage of aa ident ity and homology between HIV and FIV proteins are shown, with the three most conserved HIV and FIV proteins bolded. c Reference (REF).
44 Table 1 6. CTL epitope prediction tools Name Website Developer REF a CTLPred http://www.imtech.res.in/raghava/ctlpred/ India  NetCTL http://www.cbs.dtu.dk/services/NetCTL/ Denmark  NetCTLpan http://www.cbs.dtu.dk/services/NetCTLpan/ Denmark  a Reference (REF).
45 Table 1 7. Best defined CTL epitopes on HIV integrase Epitope Position on HXB2 HLA 1 LPPIVA KEI 28 36 B42 2 THLEGKIIL 66 74 B*1510 3 STTVKAACWW 123 132 B57 4 IQQEFGIPY 135 143 B*1503 5 VRDQAEHL 165 172 Cw18 6 KTAVQMAVF 173 181 B*5701 7 AVFIHNFKRK 179 188 A*0301 A*1101 8 FKRKGGIGGY 185 194 B*1503 9 KRKGGIGGY 186 194 B*2705 10 IIATDIQTK 2 03 211 A*1101 11 KIQNFRVYY 219 227 A*3002 12 VPRRKAKII 260 268 B42 13 RKAKIIRDY 263 271 B*1503 Adapted from LANL (http://www.hiv.lanl.gov/content/immunology/tables/optimal_ctl_summary.html) which was last updated on 2009 08 31. The best defined CTL epi specific HLA class I allele has been demonstrated with strong certainty and are judged to be at their optimal length.
46 Table 1 8. HIV 1 integrase CTL epitopes and direct FIV counterparts Allele (supertype) Vi rus Epitopes a Iden. b Hom. b IEDB Prediction: Binding Allele (nM value) c Supertype ( #alleles) NetCTL Super type B*1510 (B39) HIV THLEGKIIL B*3901(9); B*1501(425) B39(2) B39 SIV THLEGKIII 78 100 B*3901(44) B39(1) B39 FIV THFNGKIII 56 78 B*3901(6 4); B*1501(373) B39(2) B39 A*0301 (A3) A*1101 (A3) HIV MAVFIHNFK A*0301 (363); A*1101 (20) A3(5); A1(1) A3 SIV MAVHCMNFK 67 67 A*0301(174); A*1101(25) A3(4); A1(1) A3 FIV LALYCLNFK 44 78 A*3001(113); A*1101(55) A3(3); A1(1) A3 B42 (B7) HIV VPRR KAKII B*0702(43); B*0801(53) B7(1); B8(1) B7; B8 SIV VPRRKAKII 100 100 B*0702(43); B*0801(53) B7(1); B8(1) B7; B8 FIV VPRRHIRRV 44 67 B*0702(20); B*0801(124) B7(1); B8(1) B7; B8 A*1101 (A3) HIV IIATDIQTK d A*1101(404); A*6801(204) A3( 3) A3 SIV ILATDIQTT 78 89 A*0250(10) A2(5) None FIV QESLRIQDY 22 33 B*4402(88) B44(3) None FIV IVAEEIKRK e 44 78 A*1101(338); A*6801(206) A3(2) A3 a The HIV epitope sequences are from the LANL list of the best defined CTL epitopes for HIV integrase. The SIV counterpart sequences are derived from LANL SIVmm239 and the FIV counterpart sequences are derived from GenBank (ABD16378) after aa alignment with HXB2 sequence. b The identity (iden.) and homology (hom.) values were obtained using EMBOSS Stretch er Pairwise Sequence Alignment (http://www.ebi.ac.uk/Tools/psa/emboss_stretcher/). c MHC binding for HIV, SIV and FIV counterpart epitopes were predicted using the Immune Epitope Database (IEDB) MHC class I binding prediction tool (http://tools.immuneepi tope.org/analyze/html/mhc_binding.html). The matching binding alleles are shown along with their b inding affinity values (nM) which are derived from the Artificial Neural Network (ANN) analysis, where lower values represent higher binding affinity and pot ential for CD8+ T cell activity. The total numbers of binding alleles with affinity below 500 nM are shown in parenthesis next to the supertypes d HIV epitope with non matching SIV and FIV direct counterparts. e FIV epitope is an indirect counterpart with matching alleles to the HIV epitope without any direct counterpart
47 Figure 1 1. MHC class I and MHC class II molecules Also called HLA class I and HLA class II th ese are glycoproteins coded by genes located on chromosome 6. Their role is to prese nt pepti des (self or non self) on the cell surface and mediate interactions between immune cells and other cells in the body. MHC class I is found on all nucleated cells while MHC class II is found on specialized cells called antigen presenting cells (APCs) CTLs expressing the CD8 molecule with their TCR can only bind the MHC class I, while T helper cells that express the CD4 molecule with their TCR can only bind the MHC class II. MHC class I which is made up of three domains: ma i n resting on a non microglobulin), can presen t peptides of 8 11 aa long. MHC class II is made up of two domains They can present peptides of 11 30 aa in size
48 Figure 1 2. NetCTL 1.2 prediction of HIV, SIV, and FIV CTL epitopes NetCTL 1.2 which is based on proteosomic C terminal cleavage, TAP transport efficiency, and epitope binding to MHC class I alleles, was used to predict CTL epitopes shown by HLA supertypes (http://www.cbs.dtu. dk/ ser vices/NetCTL/). A) Each bar represents the number of predict ed epitopes for a particular HLA supertype: HIV (red), FIV (white), and SIV (blue ) after analysis of the full length integrase sequence from each virus. The predicted CTL epitopes were comp ared and the conserved epitopes between the viruses were identified based on aa position an d same predicted HLA supertype. B) Each bar represents the number of common epitopes between HIV SIV (red), HIV FIV (yellow), and HIV SIV FIV (green ) for a particula r HLA supertype
49 Figure 1 3 Possible location of counterpart e pitopes HIV proteins (A, B) aligned to FIV proteins (A, B) showing four HIV epitopes (h1, h2, h3, h4) and three FIV epitopes (f1, f2, f3) with arrows indicating the location of the direct counterpart (arrow a) and indirect counterpart epitopes (arrows b, and c).
50 CHAPTER 2 MATERIALS AND METHOD S Study Population A cohort of HIV 1 positive subjects from the University of California at San Francisco (UCSF), the University of South Florida (US F), and the University of Florida (UF) Shands Hospital in Jacksonville, was divided int o three groups according to time of infection and anti retroviral therapy (ART) status (Table 2 1). Cell counts and HIV 1 RNA levels in the plasma were determined by UCS F Medical Center Clinical Laboratories and Shand s Jacksonville B lood samples from ten HIV sero negative adult s w ere provided by LifeSouth Community Blood Centers (Gainesville, FL) or from randomly selected volunteers at the Universit y of Florida (UF). HLA typing was performed by the Shands Medica l Laboratory in Gainesville, FL. Ethics Statement These studies received the approval of the Institutional Review Board at UF. All blood donors provided a written informed consent for their participation in the stu dies HIV 1 and FIV Peptide Pools HIV 1 subtype B (UCD 1) overlapping peptides from the reverse transcriptase (RT) were produced using LANL PepGen Generator website and the high hydrophilic peptides were used. FIV and additional HIV 1 RT peptides were prod uced by RS Synthesis LLC (Louisville, KY). Each peptide pool consisted of four to six consecutive overlapping peptides (11 16 aa in le ngth) with an overlap of 8 11 aa. HIV and FIV RT peptides were each grouped in a total of 21 pools (pools 18 21 represent the RNAse section) representing counterparts of eac h other in term of aa position. Each pool contained 4 5 peptides. Similarly for the core capsid proteins, 18 pools of 3 4
51 overlapping peptides (8 12 aa overlap) were made for HIV and 17 pools (3 4 overlapp ing peptides) for FIV, because HIV p24 is slightly larger than FIV p24. Therefore a switch occurs after pools six for Fp7 which counterpart l i e s between Hp7 and Hp8, with subsequent alignment of Fp8 with Hp9 counterpart, and so on. Human ELISpot Analyses ELISpots protocol (R&D Systems, Minneapolis, MN) a s previously described [ 172 ]. Briefly, 1.0 2.5 x 10 5 C, 5% CO 2 for 18 24 hours. Th e assay medium consisted of AIM V medium (GIBCO, Grand Island, NY), with 10% heat inactivated (56C, 30 min) human serum (single HIV analyzed with an ELISpot reade r (MVS Pacific, LLC, Minneapolis MN) and adjusted to spot forming units (SFU) per 10 6 cells, after subtraction of the average media controls. The HIV 1 seronegative subjects had n 0 SFU) to HIV and FIV peptide pools. A posit ive response threshold of 7 0 SFU was used in the current studies. CFSE P roliferation Analysis Carlsbad, CA) protocol and processed as previously described [1 73 ]. Briefly, 2.0 5.0 x 1 0 5 PBMCs were stimulated at 37 C, 5% CO 2 for 5 days with 15 30 g of peptides. 96 or 48 well plates were used in a total volume of 200 L or 600 L respectively, of AIM V medium (GIBCO, Grand Island, NY), with 10% heat inactivated (56C, 30 min) human (Mediatech, Inc.). Before flow cytometry analysis,
52 Eugene, OR) and with fluorochrome conjugated monoclonal antibodies to human CD4 (allophycocyanin (APC)), CD3 (APC H7) and CD8 (Pacific Blue) molecules, according performed on BD LSRII (BD Biosciences) and analyzed with BD FACSDiva TM Software (BD Biosciences). The media control s well were us ed to identify proliferative resp onses as the low CFSE containing cells A positive threshold of 3 % was established after subtraction of the unstimulated media control from each subject and the average value of peptide stimulated wells from the HIV sero neg ative controls. Intracellular Cytokine Staining The cells we re processed as previously described [1 74 ]. Briefly, 0.5 1.0 x 10 6 freshly isolated PBMC we re stimulated with peptide pools ( 15 C, 5% CO 2 for 6 h in a 96 well plate, with transport inhibitor. Cells were stained with the LIVE/DEAD fixable yellow dye (Invitrogen, Eugene, OR). The following monoclonal antibodies to human cytokines and conjugated to fluorochomes were used: APC H7 to CD3 (clone SK7); BD Horizon V450 to CD4 (clone RP A T4); Qdot to CD8 (clone 3B5); PerCP to perforin (clone B D48); Alexa Fl.700 to granzyme B (clone GB11) and PE to granzyme A (clone CB9). All antibodies were purchased from BD Biosciences, except PerCP P erforin which was purchased from Abcam ( Boston, MA). The flow cytometry data is collected using BD LSRII and analyzed with the BD FACSDiva TM software. Tools Statistical analyses were performed using GraphPad Prism 5. The figures and graphs were made using GraphPad, Excel or PowerPoint.
53 Table 2 1. Population characteristics a HIV + subjects from UF at Jacksonville (J), UCSF (SF), and University of South Florida at Tampa (T ); normal blood from blood ban k (NB); normal blood from UF (N). b Number of years of HIV infection or in months (mo). c Virus load shown as copies/mL; undetectable at either 50 or 75. d NA: not available. Subject started therapy during the course of the study. Group Subject a Age Gender Race HIV b CD4/L CD8/L Viral Load c LTS/ART J01 25 F Black 11 699 935 Undetectable J10 35 F Black 12 897 659 Undetectable J11 21 F Black 21 564 829 932 J14 47 M Hispanic 25 722 142 1 6790 SF01 42 M White 16 1021 996 Undetectable SF02 44 M White 12 528 394 Undetectable SF03 59 M White 24 567 1040 5500 SF 19 40 M White 12 292 556 40000 SF08 48 M White 31 529 920 3401 SF17 53 M White 8 374 1037 2000 SF23 36 M White 7 7 84 1018 3160 SF24 47 M White 8 675 213 Undetectable ST/ART T01 19 F Hispanic <1 391 583 13400 T02 28 M White <1 1280 1375 Undetectable J02 50 F Black 9mo 639 1248 710 J03 27 F Black/Hispanic 8mo 368 1254 25700 J04 22 M Black 6mo 537 1907 1740 J05 32 M Black 2mo 384 2202 691 J06 28 M White 6mo 501 1110 134000 J07 26 F Black 4mo 448 1306 5120 J08 19 F Black/Hispanic 9mo 323 932 3040 J09 27 M White 2mo 482 882 109168 J12 26 M Pacific Islander 5mo 352 688 405000 J13 41 F Black 2mo 513 632 22100 ART + SF04 55 M White 28 540 864 Undetectable SF07 65 M White 25 610 2170 Undetectable SF20 53 M White 23 76 1172 Undetectable SF22 48 M White 13 1205 517 Undetectable SF16 50 M White 8 827 1018 Undetectable SF18 38 M White 4 1082 1 298 1500 J15 56 F Black 17 291 1101 Undetectable J16 48 F White 21 1250 NA d Undetectable HIV NB1 NA d M Unknown NB2 NA d F Unknown NB3 NA d F Unknown N1 58 F Asian N2 27 M Black N3 27 F Hispanic N4 24 M Asian N5 36 M Black N6 19 F White N7 27 F Black/Hispanic
54 CHAPTER 3 CROSS R EACTIVITY OF HIV 1 INFECTED SUBJECTS TO EVOLUTIONARY CONSERVED REVERSE TRANSCRIPTASE EPITOPES Background The commercial release of an effective HIV 1 vaccine is not imminent even after co mpletion of four major phase IIb and III vaccine trials against HIV/A IDS [ 175] Our limited understanding about the mechanisms of vaccine protection [ 4 ] and the identity of th e protective viral epitopes [177,178] further hampers the development of an effective vaccine. Initial studies have focused on the antibody based vac cine designs with emphasis on generating broadly virus neutralizing antibodies (bNAbs) [ 179 ] Two phase III envelope (Env) based vaccine trials failed [ 2 6, 28 ] Subsequent focus was placed on the T cell based vaccines that generate protective cell mediated immunity (CMI) against global HIV 1 isolates [ 2 9 ,34 ]. The CMI responses essential for an effective vaccine are most likely to include the cytotoxic T lymphocyte (CTL) activities that destroy only HIV 1 infected cells [ 50,180]. Unlike neutralizing antibody epitopes which reside exclusively on the Env proteins, the selection of vaccine epitopes for T cell based vaccines is more difficult to achieve. A vast number of CTL epitopes can be found to span the whole length of most HIV proteins [ 166 ,172] This obje ctive is also complicated by the presence of viral epitopes that enhance infection [ 116,181 ] and by the capacity of the virus to evade antiviral immunity through mutation(s) for resistance [ 182 ] A recent phase III trial consisting of priming with gag pr g p41 gp120 canarypox vectored vaccine and boosting with Env gp120 induced both humoral immunity and CMI and conferred a modest overall efficacy [ 34 ]. However, the phase I and II vaccine trials consisting of cross subtype conserved CTL peptide epitopes sho wed minimal CMI
55 responses [ 132,136 ]. Therefore, a thorough selection of potent CTL epitopes against HIV, which are conserved among HIV 1 subtypes and do not mutate without negatively affecting viral fitness [ 183 184,185 ], is required to develop an effectiv e HIV vaccine. One approach is to select conserved, non mutable CTL epitopes on essential viral structural proteins or enzymes that also persist on the older subgenuses of the lentivirinae; these have survived the evolutionary pressure [ 21 ]. A similar ap proach was used in the initial smallpox vaccines [ 162 ]. In line with this strategy, the recognition of conserved epitopes on other lentivirus species has been made by the PBMCs from HIV 1 positive (HIV + ) humans [186 ], HIV 2 vaccinated and SIV challenged non human primates , and FIV infected cats [18 7]. The viral enzyme reverse transcriptase (RT) is one of the most conserved viral proteins by possessing one of the lowest entropy value s among all the HIV 1 proteins in the major group M HIV subtypes [ 1 61 ]. The RT proteins of HIV 1 and FIV also share the highest degree of identity and homology in their aa sequences [ 21 ]. The c urrent studies have been undertaken to identify the conserved CTL epitopes on FIV and HIV 1 RT proteins which are recognized by t he PBMCs of HIV + subjects. These studies identified three major types of CMI epitopes which induced CTL activities: those on HIV 1 but not on FIV, those evolutionarily conserved on both HIV 1 and FIV, and lastly those evolutionarily conserved on FI V but uniquely lost on HIV 1. The c urrent studies demonstrate the presence of evolutionarily conserved CTL epitopes that may be more resistant to mutation, and thus useful in the development of a highly conserved, T cell based HIV 1 vaccine.
56 Screening for Inducing Epitopes on HIV 1 and FIV RT As a first step towards identifying the CTL reactive sites on HIV 1 and FIV RT proteins, the PBMC s from HIV + subjects and HIV subjects were screened by ELISpot de pools of HIV 1 and FIV. The assay does not identify which types of cells are responsible for the responses observed. PBMCs contain NK cells, CD3 + CD4 + T helper (TH) cells, CD3 + CD4 + CTLs, and CD3 + CD8 + CTLs which can in response to viral pept ides [ 1 3 ]. In this level (70 SFU) with the PBMC s from HIV + subjects (Figure 3 1A) but none with the PBMC s from HIV subjects (data not shown) Therefore the viral specif icity of the responses is associated with HIV 1 infection. The average responder frequency of all 21 pools was 25% (range, 4 54 %) (Figure 3 2A ). Pool H11 induced the highest and the most frequent responses. Compared to the HIV pool responses, the magni t ude and the frequency of the + subjects (Fig ure 3 1B and 3 2B ). The average responder frequency of all 21 pools was 17% (range, 4 69 %) (Figure 3 2B ). A noticeable exception was obse rved with the F3 pool which induced the highest and most frequent cellular responses among the FIV pools (Fig ure 3 1B and 3 2B ) The PBMC s from only f ive subjects (SF08 J02, J08, J09, T01) respon ded to counterpart pool H3 (Figure 3 1A), and remarkably th e PBMC s from four of these subjects responded to both F3 and H3. As expected, the PBMC s of HIV subjects had no responses to the FIV pools (data not shown). Overall, HIV pools induced much higher and more frequent responses than the FIV counterparts exce pt for
57 pool F3 However, only a few HIV and FIV counterparts ( 4 5 responders detected both: H3/F3, H6/F6, H7/F7, H11/F11, H13/F13) were detected by the same individual. Screening for T cell Proliferation Epitopes on HIV 1 and FIV RT The presence of st rong T cell proliferation responses to HIV antigen(s) have been associated with lower viral load and better disease outcome in HIV + individuals [188 ] In the current studies, CD3 + CD4 + T cells (hereon CD4 + T cells) from HIV + subjects (data not shown) had le ss proliferation responses than CD3 + CD8 + T cells (CD8 + T cells) to both HIV and FIV pools (Figure 3 1C and 3 1D). The CD4 + T cells from only 5 of 26 HIV + subjects responded to at least one HIV pool, whereas 9 of 26 HIV + subjects res ponded to at least one FIV pool (data not shown). In contrast, the CD8 + T cells from 16 of 26 HIV + subjects responded to HIV pools, while 2 2 of 26 responded to FIV pools (Figure 3 1C and 3 1D). The diminished CD4 + T cell proliferation responses to HIV pools were expected since m any of the HIV + subjects had low CD4 + T cell counts and generally higher CD8 + T cell counts (Table 2 1). The most striking result was the high er magnitude and frequency of CD8 + T cell proliferation to F IV pools than to HIV pools (Figure 3 1C and 3 1D). Furthermore, the average responder frequency of all FIV pools was 17% (range, 0 5 4%) (Figure 3 2B), which is higher than the average of 10% (range, 0 24 %) observed with the HIV pools (Figure 3 2A). Only one HIV pool (H11) had a r esponder fre quency >20% (Figure 3 2A), while the re sponder frequencies of the six FIV pools (F3, F6, F7, F11 F15, F21) were >20% (Figure 3 2B). Only few HIV and FIV counterparts ( 2 4 responders detected both: H3/F3, H6/F6, H14/F14/ H15/F15, H17/F17, H19/F19) were detected by the same subject, but 42% (22 of 52) of the total positive responses to HIV pools were positive to FIV counterparts.
58 All of the above results support the view that the CD8 + T cell proliferative responses to FIV pools are more robust or possib ly more i ntact than those to HIV pools. stronger against the HIV pools than t he FIV pools (Figure 3 1 A and 3 1B ). These conflicting findings may be partially attributed to the difference in the cell types used (PBMC versu s CD8 + T cells) Moreover, 12 of 14 responders of CD8 + T cell proliferation ure 3 1B and 3 1D) These results suggest that these subjects recogniz e p eptide epitope(s) that induce both responses. Proliferation Responses to Selecte d HIV and FIV Peptide Pools Due to the ability of HIV to quickly esc ape from immunological pressure [51, 157,158 ], the PBMC s ure s 3 1A and 3 1B were retested peptide pools H6, H11, and F3. The majority of the individuals tested retained positive responses at the second time point to H6 (7 of 10 responders) and to F3 (6 of 7 responder s) but not to H11 (4 of 13 responders) (Figure 3 3 A ) A third time point taken for H6 and F3 demonstrate s the persistence of the responses to these peptide pools for 3 years (data not shown). More importantly, the persistence of the responses to F3 demo nstrates the reproducibility of this response even though no responses have be en observed to counterpart H3. The CD8 + T cell proliferation responses to F3 ( 7 of 9 responders ) persisted but not to H11 ( 3 of 5 ) and F6 (1 of 3 ) (Figure 3 3 B ) Overall, these results suggest a major loss of HIV specific proliferation responses while retaining longer responses to F3 pool.
59 Identifying the Peptide Epitope(s) o n F3 nd CD8 + T Cell Proliferation Responses The finding that 69% and 54 % of the HIV + subjects responded to pool F3 with + T cell proliferation respectively (Figure 3 2A and 3 2B ) suggest s the potential that the F3 region contain s multiple epitopes. The F3 peptide pool contains five overlapping peptides of 13 15mers (F3 1, F3 2, F3 3, F3 4, and F3 5). Ni ne of 11 F3 responders tested 1 the F3 3 peptide, and one s to individ ual peptides F3 1 and F3 4 (Figure 3 4 A ). Furthermore 4 of 10 F3 responders had rpart pool H3 in the first year. However, all three from the se H3 responders who were tested on or after the 2 nd year lost the response to H3 and had no response to any of the individual 13 15mer H3 peptides. As expected, all t en HIV control subjects had no responses to individu al peptides of both F3 and H3. observed to pool F3 was attribute d to the F3 3 peptide, and the highest responder frequency of CD8 + T cell proliferation was obser ved with F3 3 (5 o f 8 ) and slightly lower with F3 1 (3 of 5) and F3 5 (3 of 5) ( Figure 3 4 B ). F3 3 is therefore the predominant peptide that induc e Characterization of CTL Activities Induced by F3 3 Pepti de One of the most important CMI activities needed to control HIV infection are potent CTL activities [61 ] Both CD4 + CTLs and CD8 + CTLs against HIV 1 have been detected in HIV + subjects [ 5 6 ] and in HIV individuals immunized with can didate HIV 1 vaccine s [ 139 ]. Us ing the ICS approach, both CD3 + CD4 + and CD3 + CD8 + T cells expressed cytotoxin(s) ( granzyme A: GrzA; granzyme B: GrzB ; or perforin : Perf ) upon stimulation wit h pools F3, F6, H6, or H11 (Figure 3 5A to 3 5 C). 100% (11 of 11) of the
60 F3 responders had at least one cytotoxin expression in their CD4 + or CD8 + T cells similar to the 100% (8 of 8) of H11 resp onders but different from the 75% (6 of 8 ) of H6 responders and 50% (3 of 6) of F6 responders. Hence, CTL epitope(s) present on F3 and H11 were recognized by all tested, suggesting that multiple CTL epitopes may reside on each of these regions. The F3 peptide study show ed all five F3 peptides can induce GrzA, GrzB, and/or perforin in the CD4 + and/or CD8 + T cells of at least one or more HIV + subjects tested (Fig ure 3 5 D ). Based on this finding, CTL epitope(s) appear to be on all of the indiv idual F3 peptides. Determining whether H3 3 and F3 3 are Conserved among Lentiviruses T he H3 pool makes up a stretch of aa ( location: 54 88 aa on RT) that is highly conserved a mong lenti viruses as it is identical to 47% of the HIV % of the ccording to LANL QuickAlign analysis ( http://www.hiv.lanl.gov/content/sequence/QUICK_ALIGN/QuickAlign.html ). Meanwhile H3 3 has 83% and 35% aa identity with HIV 1 and SI V sequences respectively, by the same analysis system. The full sequence of H3 r egion has 69% aa identity and 80 % a a homology (with one gap) to the counterpart F3 sequence while H3 3 and F3 3 peptides have 69% identity and 75 % homology (with one gap) (Tab le 3 1 top ). Even with such + T cell proliferation responses greatly di ffered between these peptides. The 15 mer F3 3 differs from the 15 mer H3 3 used in the current study (rows 1 versus 2, Table 3 1 ) by lacking one aa D (a sp artic acid at position 4 of H3 3) and having four aa differences at F3 3 positions 5, 9, 11, and 15. The combination of D 4 insertion and changes with H3 3 corresponding aa ( T 6 K 10 V 12 and E 16 when necessary ) in to the F3 3 sequence helped identify important aa responsible for the disparity of responses observed between F3 3 and H3 3 (Table 3 1 ). The
61 insertion of D 4 in to F3 3 ( F 3 3m2 : 16mer) and substitution of G 5 for T 6 (F3 3m6: 15mer) r esulted in responses approaching F3 3 whereas the removal of V 16 from F 3 3m 2 resulting in the F 3 3m1 15mer in proliferation responses. Fur thermore, different 15mers of F3 3 obtained by a single aa change at positions 9 (M 9 10 ; F 3 3m5), 11 (I 11 12 ; F 3 3m3), or 15 (V 15 16 ; + T cell proliferation re sponses. It is n ote worthy that none of the modifications of H3 3 an d the peptides tested in Table 3 1 including the HERV respons es in the PBMC or the T cells from HIV control subjects. Peptide F3 3 has high degrees of aa identity to those of ungulate lentiviruses (93%, caprine arthritis encephalitis virus [CAEV] and Maedi Visna virus [MVV]) and slightly less identity to those of h uman endogenous retroviruses (HERV, 80%), HI V 1 (69%) with one gap and SIV (69 %) with one gap (Table 3 1 top). Thus, the F3 3 sequence is greatly conserved among lentiviruses. The u ngulate peptide counterpart of F3 3, but not HERV s in the PBMC from one F3 3 responder (Table 3 1 top). Moreover, the CD8 + T cells from one F3 3 responder proliferated in response to HERV K peptide. The above results demo nstrate that the F3 3 sequence is an evolutionarily conserved epitope(s) that induce persistent CMI responses, including strong CTL activity, even when the responses to counterpart H3 3 are lost.
62 Table 3 1. Variation of H3 3/F3 3 aa sequences and immunolo gical responses Virus (Subtype) a 9mer or 15mer Sequence b Average SFU o [range] (positive/total) cd % CD8 + T Proliferation [range] (positive/total) cd Evolutionary epitopes: HIV 1 H3 3 KKKdStKWRkLvDFR 0  (0/3) 0  (0/1) FIV F3 3 KKK SGKWRMLIDFRV 536 [105 2500] (13/13) 5.4 [0 15] (6/9) HIV 1 (C) KKK StKWRkLvDF Re 7 [0 37] (0/9) 0  (0/5) HIV 1 (A,B,C,D, 01_AE) KKn StKWRkLvDFRe 0  (0/9) 0.9 [0 4] (1/5) SIVcpz Pts KKKdStKWRkLvDFRe e ND ND CAEV & MVV KKK SGKWRMLIDFRe 28 [0 100] (1/9) 0.2 [0 0.8] (0/5) HERV K KKK SGKWRMLtDlRa 13 [0 60] (0/9) 0 .5 [0 3] (1/5) Modifications of H3 3: f HIV 1 H3 3 KKKdStKWRkLvDFR 0  (0/3) 0  (0/1) FIV F 3 3m1 KKKdSGKWRMLIDFR 11 [0 45] (0/9) 0.7 [0 3.2] (1/5) FIV F 3 3m2 KKKdSGKWRMLIDFRV e 280 [0 1208] (7/9) 2.0 [0 5.6] (2/5) FIV F 3 3m3 KKK SGKWRMLvDFR V 7 [0 22] (0/9) 0  (0/5) FIV F 3 3m4 KKK StKWRkLIDFRV 9 [0 60] (0/9) 0.2 [0 0.9] (0/5) FIV F 3 3m5 KKK SGKWRkLIDFRV 20 [0 52] (0/9) 0.3 [0 1.6] (0/5) FIV F 3 3m6 KKK StKWRMLIDFRV 251 [20 1254] (7/9) 7.0 [0 29.1] (2/5) FIV F3 3 KKK SGKWRMLIDFRV 53 6 [105 2500] (13/13) 5.4 [0 15] (6/9) Epitopes on F3 3: g FIV F3 3 (KV15) KKK SGKWRMLIDFRV 536 [105 2500] (13/13) 5.4 [0 15] (6/9) FIV 3 3 (WV9) WRMLIDFRV 278 [0 1295] (8/11) 1.3 [0 4.2] (1/6) FIV 3 3 (KR9) KWRMLI DFR 20 [0 70] (1/9) 0  (0/6) a Genbank numbers as follows: HIV 1 H3 3 (AAB04503); FIV F3 3 (AAB18889); HIV 1 (C) (FJ595343); HIV 1 (A,B,C,D, 01_AE) (AJ313415, HM035584, HQ012309, HQ586068, HE590997); SIVcpz Pts (ACM63211); CAEV (AAB02107); MVV (CAC4454 3); HERV K (ABA28284). b Lower case letter for aa different from FIV F3 3. Glutamate (E) is added at position 15 to obtain 15mer. Many HIV 1 strains have E immediately after the carboxyl end of H3 3. c Used only responders from Figures 1 and 3; range responses over total tested (positive/total). d The total number of subjects is small for some groups because the data shows only the F3 or H3 responders who are still positive to the corresponding pool during t he second or third time point. e Only 16mer sequence s f Six modifications of 15 16mer F3 3 sequences (F3 3m1 to F3 3m6) to resemble H 3 3 sequence Replacing V15 with E15 in the four modifications F3 3m3 to F 3 3m6 resulted in a proliferation responses (data not shown). g Sequence designation shown in parenthesis with the first and the last aa followed by the number of aa.
63 Figure 3 ELISpot (1A and 1B, n=32) and CD3 + CD8 + T cell proliferation (1C and 1D, n=26) responses to overlapping peptide pools of HIV RT (H1 H21; 1A and 1C) and FIV RT (F1 F21; 1B and 1D) are shown. The HIV + subjects (panel A insert for all panels) consisted of long term survivor s (LTS) who have had an HIV infection for over 10 years without antiretroviral therapy (ART) (LTS/ART ; black bar); recently diagnosed, short term infection without ART (ST/ART ; grey bar); and those on ART at various duration of infection (ART + red bar). Each bar represents a positive response by an individual with a threshold of 70 spot forming units (SFU) per 10 6 PBMC for ELISpot or threshold of 3% CFSE low for CD3 + CD8 + T cell proliferation. The HIV control subjects (n=10) had no responses (data not s hown). All responses below the positive threshold are not shown.
65 Figure 3 2. Frequency of positive HIV RT (H) and FI V RT (F) peptide pool responses Immunological responses a re assessed for 32 HIV + ELISp ot assay (black bars) and for 26 of these individuals in the CFSE proliferation assay (grey bars) from Figure 3 1 Positive r esponse thresholds were set at ( 7 0 spot forming units per million PBMC after subtraction of t he media control (ELISpot) and ubtraction of the media control (proliferation)) Each bar represents the frequency of positive responses observed A) to each H pool (H1 H21), and B) to each F pool (F1 F21) Proliferation (Prolif.).
66 Figure 3 3 RT peptide pools. and B) the CD8 + T cell proliferation responses of HIV + subjects who responded at the first time point (t1) and second time point (t2, at l east 1 year later) are shown for peptide pool F3 (both analyses), H11 (both analyses), p value on top of each peptide pool group tests whether the results from t1 are statistically different from those from t2.
67 Figure 3 4 F3 peptide epitopes recognized by F3 responders. Peptide pool F3 consists of five overlapping 13 15mer peptides spanning from amino to carboxy l terminus (F3 1, F3 2, F3 3, F3 4, and F3 5). n=10) and B) CD8 + T cell prol iferation ( n=8) responses of F3 responders to each of these peptides are shown along with responses to F3 pool. F3 responders consist of those with long term infection but not on ART (LTS/ART ; black bar); with short term infection and not on ART (ST/ART ; grey ba r); and those on ART with various duration of infection (ART + ; red bar). All responses below positive thresholds are not shown.
68 Figure 3 5. Characterization of CTL epitopes on F3, H6, F6, and H11 pools. ICS analysis for A) granzyme B (GrzB); B) granzym e A (GrzA); and C) perforin (Perf) are shown for CD8 + T cells (left column) and CD4 + T cells (right column) from selected HIV + responders of designated peptide pools (A C), using a positive threshold of 1%. The HIV + subjects (A C) consists of the following individuals that have long term infection without ART (LTS/ART ; full black circle ); recently diagnosed, short term infection without ART (ST/ART ; full grey circle ); and those on ART with various duration of infection (ART + ; open circle ). D) Six F3 pool responders (five LTS/ART and one ART + 15mer F3 peptides (D). Only three subjects were the same as those from above (A C) but from blood collected at different time point.
70 CH APTER 4 CONSERVED VACCINE EPITOPES ON H IV 1 AND FIV P 24 RECOGNIZED BY HIV 1 INFECTED SUBJECTS esponses The PBMC from 3 1 HIV + length of the HIV 1 p24 peptide pool wi th a total of 183 responses (Figure 4 1A), whereas no responses were observed with the PBMC from HIV cont rol subjects (data not shown). The highest responder frequencies were observed to H p 3 (19 of 31; 61%) and H p 10 (16 of 31; 52%) followed by H p 2 (13 of 31; 42%) and H p 15 (13 of 31; 42%), and then H p 7, H p 11, H p 12, and H p 14 (latt er four pools, 12 of 31; 39%). Those peptide pools with large numbers of responders (e.g., 42 61%) are likely to have multiple CMI epitopes. Due to the robust responses to many HIV pools, the most important epitopes are not clearly identifiable On the other hand with the exception of peptide pool F p 14, the PBMC from the HIV + frequencies to FIV p24 peptide pools with a total of 67 responses (Fig ure 4 1B). The highest responder frequencies were observed to F p 14 (1 3 of 31; 42 %) followed by F p 13 (6 of 31; 19%) and then F p 7, F p 10, and F p 11 (latter three pools, 5 of 31; 16%). The PBMC from eight HIV + subjects responded to both F p 14 a nd its HIV counterpart pool H p 15, which suggested the presence of epitope(s) common between these counterpart pools and thus the detection of evolutionarily conserved epitope(s). Conserved CMI Epitopes Based on T cell P roliferation R esponses As another app roach to identify protective CMI epitopes, CFSE proliferation responses of both CD3 + CD4 + and CD3 + CD8 + T cells were analyzed using the same ove rlapping HIV and FIV p24 pools. The CD8 + T cells of the HIV + subjects proliferated more frequently at a higher mag nitude to HIV p24 peptide pools (Fig ure 4 1C) than the
71 CD4 + T cells (data not shown). A total of 41 CD8 + T cell responses were detected compared to the total of 13 CD4 + T cell responses. In contrast, 32 CD8 + T cell proliferation responses to F IV p24 pools were detected (Figure 4 1D) compared to nine CD4 + T cell proliferati on responses (data not shown). The highest frequency of the CD8 + T cell proliferation responses were observed to H p 1 (6 of 26; 23%), H p10 (8 of 26; 31 %), and H p 15 (6 of 26; 23%) followed b y H p 9 (4 of 26; 15%) and H p 12 (4 of 26; 15%) (Figure 4 1C). Strikingly, substantial responses were observed to F p 9 (11 of 26; 42%) and then to F p 14 (6 of 26; 23%) (Figure 4 1D). F p 9 and its counterpart H p 10 had CD8 + T cells from six HIV + sub jects respondin g to both pools. The lower numbers of epitopes for CD8 + T cell proliferation. Overall, CD8 + T cell proliferation res ponses even to the HIV pools (42 total responses) were le ss prominent when compared to the robust to FIV p ools (67 total responses). esponses to F p 14 and CD8 + T cell P roliferation to F p 9 In the above studie s, cells from a sizable number of HIV + subjects developed p 14 and strong CD8 + T cell proliferation responses to F p9. The PBMC of 8 of 10 HIV + subjects who initially responded to F p responses to F p 14 after the 2 to 3 year period between tests (Figure 4 2A, left), while the PBMC of 6 of 8 initial responders to H p 15, the counterpart of F p 14, remained responsive to H p 15 (Figure 4 2A, right). Likewise the CD8 + T cells from 6 of 7 initial F p 9 responders retained prolifera tion responses to F p 9 after a 2 to 3 year period (Figure 4 2B, left). In contrast only 2 of 5 initial responders to H p 10, the counterpart for F p 9,
72 retained responses to H p 10 for 2 3 years (Figure 4 2B, right). The responses to pools F p 9 and F p 14 persisted for over 2 years and were thus reproducible. Identifying the E pitope(s) on F p 9 and F p 14 Regions that Induce CMI R esponses Three to four overlapping 13 15mer peptides constitute each of the peptide pools F p 9 (F p 9 1, F p 9 2, F p 9 3) and F p 14 (F p 14 1, F p 14 2 F p 14 3, F p 14 4). These shorter peptides and the 15mer peptides from a counterpart pool H p 15 were used to identify CMI epitopes + T cell proliferation. F p 14 and H p 15 have aa sequence similari ty of 61% and identity of 35%. Based on aa sequence alignment analysis, the approximate counterpart for H p 15 1 and H p 15 2 are F p 14 1 and F p 14 2, respectively. The H p 15 3 is a counterpart region that overlaps both F p 14 3 and F p 14 4. The m to F p 14 peptides by F p 14 pool responders were detected to the F p14 3 peptide (6 of 9 responders) followed by peptides F p 14 1 (3 of 9) and F p 14 4 (3 of 9) (Figure 4 3 p 15 peptides by the H p 15 responders were to H p15 1 (7 of 9 ) and some to H p15 3 (5 of 9 ) (Figure 4 3B). p 14 3 and H p 15 1, but these peptides are not counterpa rt peptides based on aa sequence analysis. Consequently, these peptides are most likely expressing epitopes that are not common. However, three LTS subjects responded to both F p 14 3 and counterpart H p 15 3. This observation may suggest the potential existe nce of a CMI epitope conserved between the two peptides. The majority of the CD8 + T cell proliferation responses made by the F p 9 responders were directed to F p 9 3 (6 of 7) and few to F p 9 2 (3 of 7) (Figure 4 3C). These CD8 + T cell responses were also det ected at a low magnitude to all five H p 10 peptides by different combination of subjects (3 of 4 subjects for e ach peptide) (data not
73 shown). The counterpart comparison between F p 9 peptides and H p 10 peptides is not shown and is not discussed mainly because of the low aa sequence similarity between the F p 9 and H p 10 regions (16% identity; 36 % similarity). Characterization of CTL Activities I nduced by F p 9 and F p 14 P ools An increase in GrzB expression was most consistently observed to all three peptide pools (Fp 9, Fp14, and Hp15) in both CD8 + and CD4 + T cells (figure 4 4A) The responder frequency rates of CD8 + T cells were 71% (5 of 7) for all of these pools. In contrast, the responder frequency rates of CD4 + T cells were 43% (3 of 7) for Fp9, 71% (5 of 7) for F p14, and 57% (4 of 7) for Hp15. Few of the subjects who did not have GrzB often had either an increase in GrzA or perforin expression (Figure 4 4) The majority of these peptides induce d one or more cytotoxin expression and thus, these peptides express ed CTL epitope(s). Compared to the cytotoxin responses to RT peptide pools in the previous chapter, the cytotoxin responses to p24 peptide pools were much lower in numbers and for some cytototoxin s also in magnitude (Figure 4 4). This study used a lower numbe r of specific responders tested (n=7 for all p24 pools), while with the except ion of one RT pool (F6, n=6), all remaining RT pools had either 8 or 11 responders. Even with this taken into consideration, the numbers of CD8 + T cell responders for GrzA and CD 4 + T cell responses for perforin were extremely low in numbers Since similar observation for GrzA in CD8 + T cells was detected with RT pools, it may be sp eculated that this low response may possibly be attributed to the intrinsic inability of the CD8 + T cells from HIV + subjects to produce Gr z A as also indicated by the lack of responses to mitogenic stimulation.
74 Figure 4 1. ELISpot (1A and 1B, n=31) and CD3 + CD8 + T cell proliferation (1C and 1D, n=26) responses to overlapping peptide pools of HIV p24 (Hp1 Hp18; 1A and 1C) and FIV p24 (Fp1 Fp17; 1B and 1D) are shown. The HIV + subjects (panel A insert for all panels) consisted of long term survivors (LTS) who have had HIV an infection for over 10 years without antiretroviral therapy (ART) (LTS/ART ; black bar); recently diagnosed, short term infection w ithout ART (ST/ART ; grey bar); and those on ART at various duration of infection (ART + red bar). Each bar represents a positive response by an individual with a threshold of 70 spot forming units (SFU) per 10 6 PBMC for ELISpot or threshold of 3% CFSE low for CD3 + CD8 + T cell proliferation. The HIV control subjects (n=10) had no responses (data not shown). All responses below the positive threshold are not shown.
75 Figure 4 2 p24 peptide pools The + T cell proliferation (B) responses of HIV + subjects who responded at the first time point (t1) and second time point (t2, a2 to 3 year s later) are shown for peptide pool F p p 9 (proliferation). The subjects from the different pools presented are not the same in most cases. The full black circles represent the long term survivors not on ART; the full gray squares represent the short term diagnosed not on ART; and the open triangles represent the subjects on A RT.
76 Figure 4 3. Fp and H p peptide epitopes recognized by pool responders. Peptide pool F p 14 consists of four overlapping 13 15mer peptides spanning from amino to carboxy l terminal (F p 14 1, F p 14 2, F p 14 3, F p14 4 ), while pools H p 15 (H p 15 1, H p 15 2, H p 15 3 ) and F p 9 (F p 9 1, F p 9 2, F p 9 3 ) consist of three overlapping 13 15mer p 14 peptides (A; n=9) and to H p 15 peptides (B; n=9 ) and CD8 + T cell proliferation responses to F p 9 peptides (C; n=7) are shown. The responders consist of those with long term infection but not on ART (LTS/ART ; black bar); with short term infection and not on ART (ST/ART ; grey bar); and those on ART with various duration of infection (ART + ; red bar). Seven of the HIV + subjects (four LTS/ART one ST/ A RT and two ART + p 14 and H p 15 were tested for both pools, while two ST/ART subjects for F p 14 peptides and one each of the LTS/ART or ST/ART subject for H p 15 peptides were tested for only one pool (F p 14 or H p 15). All responses below positive thresholds are not shown. Those subjects who responded to both Fp14 3 and Hp15 3 are shown with above the bars.
77 Figure 4 4. Characterization of CTL epitopes on F p 9 Fp14, and H p 15 pools. ICS analysis for A) granzyme B (GrzB); B) granzyme A (GrzA); and C) perforin (Perf) are shown for CD8 + T cells (left column) and CD4 + T cells (right column) from selected HIV + responders of designated peptide pools (A C), using a positive threshold of 1%. The HIV + subjects (A C) consists of the following in dividuals that have long term infection without ART (LTS/ART ; full black ); recently diagnosed, short term infection without ART (ST/ART ; full grey ); and those on ART with various duration of infection (ART + ; open ). Seven responders are shown for each cyt otoxin, and only three responders overlapped among all three peptide pools.
78 CHAPTER 5 DISCUSSION Identification of CMI R esponses on HIV and FIV RT The CMI responses made by the HIV + subjects to FIV and HIV RT peptides or peptide pools resulted in three ma jor observations. First, the CD8 + T cell proliferation responses to FIV pools are more robust with higher frequency of responders than those in duced by HIV pools (Figure 3 2A and 3 2B ). This observation was unexpected since were observed with HIV pools than with FIV pools. These proliferation responses to FIV pools, especially to F3, persisted over a longer time period than those responses to HIV pools. Thus, the few aa differences between the two viruses may be sufficient enough for the CD8 + T cells to only recognize the F3 but not the H3. Another possibility is the depletion or anergy of the H3 responsive CD8 + T cells caused by the constant exposure to HIV, similar to T cell exhaustion s as previously described [ 113,18 9]. The first possibility was tested and the results showed that three of the 5 aa differences between H3 3 and F3 3 were independently important for the divergent responses observed There was no statistically significant difference between the population groups (LTS/ART ST/ART ART + ) in terms of cell counts. T here was no particular pool inducing responses uniquely in subjects f rom one group and not the other groups However, there was a trend at the individual level based on sample origins H6 pool tend ed to be the most dominant pool in terms of magnitude and frequency for samples originating fr om San Francisco, while H11 tend ed to be most dominant for samples originating from Jacksonville. The majority of samples from San Francisco were LTS/ART while t he majority of samples from Jacksonville were ST/ART
79 The robust CD8 + T cell responses by the HIV + subjects to FIV peptide pools suggest the possibility that these peptide regions contain evolutionarily conserved epitopes. Importantly, 42% (22 of 52 ) of the total positive CD8 + T cell proliferation responses and 23% (39 of 166 also positive for their counterpart FIV pools. Hence, the conserved CMI epitopes are easier to screen by CD8 + T cell prolife Note that the contribution of NK cell responses was not measured in these studies. + T cell proliferation responses to the F3 pool whic h had more responders than any HIV pool the PBMC from a large number ( 69 %) of HIV + subjects and CD8 + T proliferation res ponses in a substantial number (54 %) of responders. These results suggest the presence of multiple CD8 + T cell epitopes in the F3 region. One to three F3 CD8 + T proliferation responses to F3 1 and F3 4, and both peptides induced CTL activities In fact, LANL analysis of the H3 counterpart shows three CTL epitopes (NTPVFAIKK, NK9; NTPVFAIKKK, NK10; and KLVDFRELNK, KK10). The NK9 and NK10 sequences are identical between FIV and HIV 1 and are found at the carboxy l end of both 13mer peptides F3 1 and H3 1. F3 1 only differs from H3 1 by having tryptophan (W 3 ) instead of tyrosine (Y 3 ) at position 3. This finding suggests that this single aa difference resulted in CD8 + T proliferation to F3 1 but not to H3 1. In the case of KK10, this epitope resides o n H3 4 and differs by three aa from its direct counterpart on F3 4 ( mLiDFRvLN K (MK1 0); differen t aa in lower case).
80 The third major observation was the robust tested) and CD8 + T proliferation (63 %, 5 of 8 ) responses to the 15 mer peptide F3 3 (Figure 3 4 ). These unusually high frequencies of responders to F3 3 peptide raised a question on whether more than one CMI epitopes reside on F3 3. In this regard, the current studies identified three CMI epitopes on F3 3, which were not previously described in LANL: KKKSGKWRMLIDFRV (KV15), WRMLIDFRV (WV9), and K WRMLIDFR (KR9) (Table 3 1 bottom ). These epitopes induce CTL responses to F3 3, and consequently they are CTL epitopes. Furthermore, these epitopes are closely related in sequence and evolution to ungulate lentiviruses and HERV K, indicating that F3 3 ep itopes are also evolutionarily conserved. Other studies have shown the higher expression of HERV proteins and higher responses to HERV specific peptides in HIV + subjects than in HIV subjects [19 0]. Those responses are associated with virus control [19 1]. Furthermore our results suggest that the responses to evolutionarily conserved F3 epitopes which overlap with HERV epitopes (with a few aa differences) do not induce responses in normal HIV subjects. This cross recognition of the F3 3 epitope(s) by the H IV + subjects demonstrates the polyfunctionality of the T + T proliferation, and + T proliferation and cytotoxins. Epitopes able to induce p olyfunctional activities are consider ed to be important for an effective HIV vaccine [100 ]. Although the current studies have had minimal focus on CD4 + T cell responses, the two F3 responders of CD4 + T proliferation also expressed CTL responses to F3 3 (data not shown), and the CD4 + T cells from substantial numbers of F3 responders had CTL activities to pool F3 (Figure 3 5 ). Given that the prophylactic
81 vaccine is generally administered to HIV nave subjects with normal CD4 + T cell immunity, the importance of CD4 + T cell responses to F3 3 can not be ignored when identifying CTL epitopes for an HIV vaccine [ 63,64 ] Th erefore the vaccine epitopes that induce both anti HIV CD8 + CTLs and CD4 + CTLs are likely to be important for an effective vaccin e In conclusion, the use of FIV RT peptide pools has identified evolutionarily conserved CTL epitopes which may be important as CTL epitopes for a preventive HIV vaccine or for post infection strategies Evolutionarily C onserved CTL Epitopes on HIV 1 and FIV p24 P roteins Unlike the RT results with one p ool F3 having the most CMI responses, the cells from HIV + subjects detected two potential CMI epitopes on pools Fp9 and Fp14 based proliferation analysis systems. Pool Fp14 + T ce ll proliferation responses (Figure 4 1), whereas pool Fp9 induced strong CD8 + T cell proliferation in a large number of HIV + subjects. The responses to F p 9 and F p 14 were highly reproducible and persisted over 2 years (Figure 4 2). Both Fp14 (42 %) and Fp9 (42%) had a high responder frequency which indicated that these regions most likel y expressed multiple epitopes. The individual 13 15mer peptide analysis identified one 14mer peptide Fp14 3 to have the most responses followed by Fp14 1 (14mer) and Fp14 4 ( 13mer). The fact that Fp14 1 and Fp 14 4 overlap by only five aa suggests the potential for CMI epitopes on these peptide s to be different. NetCTL analyses for HLA class I and II also identifies different CTL/TH epitopes for these peptides (data not shown). The Fp14 3 has 71% similarity (10 of 14 aa) (4 of 14 aa, 29% identity) to the counterpart Hp15 region; while 11 aa section of the Fp14 3 (carboxyl end) has 82% similarity (9 of 11 aa) (3 of 11 aa, 27% ident ity) to the
82 counterpart Hp15 3. This sequence co mparison suggests the possibility for an epitope conserved between Fp14 3 and Hp15 3 as supported by the three LTS subjects r esponding to both peptides (Figure 4 3A and 4 3 B). This concept is supported by the observation that the positive expressions of Gr zA, GrzB, and perforin to Fp14 3 correlating with positive expressions of the same cytotoxins in responses to Hp15 3 (6 of 7 or 86% positive responses). Both Fp14 3 and Hp15 3 induce the same cytotoxins especially in CD8 + T cells and therefore, they most l ikely contain common CD8 + CTL epitope s Thus, F p 14 was used to select evolutionarily conserved CTL epitope on H p 15. The carboxyl end of the F p 9 3 peptide has 58% similarity (7 of 12 aa) and 50% id entity (6 of 12 aa) to HERV K. S tudies by others show higher responses to HERV specific peptides in HIV + subjects [19 0], and such responses have bee n associated with HIV control [19 1]. This observation has led to the concept of using HERV or HERV like epitopes as targets for an HIV vaccine owing to the fact that t h ey most likely do not mutate [19 2 ] Controversy still exists on the potential of vaccine epitopes resembling HERV proteins to cause autoimmunity [ 193,194,19 5 ] However, recent studies have shown them to be safe and immunogenic with primates [19 6 ] In our s tudy, the Fp9 3 peptide was recognized by HIV + subjects but not by HIV control subjects, which supports the use of Fp9 3 for HIV 1 vaccine even though it has an evolutionarily conserved sequence simil ar to HERV. Importantly, F p 9 3 induced more CD8 + T cell s/ GrzA and CD8 + T cells/ GrzB than Fp9 pool from the Fp 9 responders (data not shown). Hence F p 9 3 also contains evolutionarily conserved CTL ep itope(s).
83 Identification and Use of Evolutionary Conserved Epitopes The current studies have demonstrated the u se of a lentivirus as a tool to identify evolutionary conserved regions across species and possibly beyond lentiviruses as sequence conservations hav e also been observed with HERV. The conserved pools identified both on the p24 s and RT s most likely conta in multiple MHC class I and class II epitopes as observed on LANL (http://www.hiv.lanl.gov/content/immunology/index). FIV responding epitopes can be considered as counterpart variants of HIV sequences and further characterized for specific binding alleles. Testing whether vaccination with an HIV epitope can induce immune responses to its FIV counterpart or vice versa is the first step towards verifying the validity of the evolutionary conserved epitope approach between the two viruses as described here for protective immunity. Whether HERV sequences are involved in HIV/AIDS autoimmunity is not clear but t he fact that those sequences are naturally present in humans (potential vaccinees) requires additional saf ety considerations The current studies have ma de a limited use of in silico analysis to identify epitopes. Some of the predicted epitopes have been tested in biological assays by IFNy ELISpot. 53% (8/15) predicted epitopes induced a positive response in the PBMCs from at least one i ndividual (data not shown). These data have shown that in silico tools is useful in combination with biological assays. The WV9 epitope with 8/1 1 positive responders (Table 3 1 ) is predicted to bind B27 alleles (NetCTL 1.2) which are associated with better outcomes of HIV in fection. In fact, HLA B27 alleles have been associated with bett er control of viral infections  and with many spondyloarthropathies [ 198 ] such as ankylosing spondylitis or psoriatic arthritis (PA) which is a autoimmune disease als o encountered in HI V patients [ 19 9 ]. It is unlikely that
84 the majority of all responding subjects share the same MHC alleles because of the low frequency of B27 in the US population. The high frequency of responses to WV9 most likely indicate s t he promiscuity of this epitope at bind ing multiple MHC alleles. Efforts to identify an tigens responsible for the link betwe en B27 (but not exclusive) and PA for example need to consider this conserved region described on the RT. Tests of HIV / PA patients along with HLA B27 allele matc hed controls should give an indication of the role of this region not onl y in autoimmunity, but also in its potential use a s part of a vaccine immunogen In addition, H3 3 lies in a conserved domain (aa 20 190 on HIV RT) a region that was identified as t he most evolutionarily conserved of the RT and prop o sed to bind nucleic acids duri ng reverse transcription [ 200 ,201 ,202 ]. T he H3 3 segment also contains drug resistant mutat ions associated with nucleotide reverse transcriptase inhibitors [20 3 ] including m utations at those aa positions that differ from the F3 3 sequence The current studies have identified some of those aa as essential for the responses observed against F3 3. The H3 3 region al so contains some of the amino acid s with possible mutations ( lik e D 1 3 V and R 15 A) that were sh own to increase the fidelity of DNA synthesis of the RT and also to affect its overall processivity [ 201,202 ]. An increased fidelity of the RT enzyme mean s fewer mutations, something that c an help the immune system focus on few er HIV variants. Therefore, understanding the role of these immunological responses during HIV infection s and treatment s can also prove helpful for future drug design s and for immunization strategies pre and post infection.
85 Overall Summary In conclusion, six major observations were made by comparing the results from HIV/FIV RT studies to those of HIV/FIV p24 studies. 1) CFSE CD8 + T cell proliferation responses to FIV RT pools were more robust than those to HIV RT pools. This pattern was the opposite from th e results observed with FIV and HIV p24 pools. 2) Both F3 pool and F 3 + T cell proliferation responses. In contrast, the induc tion of these CMI responses was distributed into two pool/peptide sets: FIV p24 Fp9 and Fp9 3 for CD8 + T cell proliferation and Fp14 and Fp14 3) Based on ICS analysis, all of these peptides contained CTL epitopes ; however as expected, the type s of cytotoxin(s) expressed differed between the pools/peptides ( F3/F3 3, Fp9/Fp9 3, Fp14/Fp14 3) and the individuals tested. 4) These CTL epitopes retained evolutionary lineage with some more evident than others by sequence comparison The fact that these epitopes were selected using FIV peptides with human PBMC furth er supports the ir evolutionary lineage. 5) Conserved CTL epitopes were found on counterpart HIV 1 prote ins such as RT H6 and p24 Hp15. 6) These CTL epitopes possess a polyfunctional activity, which is expected to be essential for an effective HIV 1 vacci ne. The a bove observations confirm that FIV peptides can be used to select evolutionarily conserved CTL epitopes with polyfunc tional activity against HIV 1. These epitopes are remnants of evolutionary lineage of both lentiviruses and retroviral family and are present on essential structural protein (s) or enzyme (s) of the virus. T hese conserved CTL epitopes are less likely to mutate (without an associated fitness cost to the virus) as supported by their existence and persistent recognition by the HIV + sub jects with various length of infection and ART status. The a bove observations also
86 demons trate the use of FIV peptides for the select ion of evolutionarily conserved CTL epitopes that are on HIV 1 but not on FIV; those evolutionarily conserved on both HIV 1 and FIV; and finally those evolutionarily conserved on F IV but uniquely lost on HIV 1. Overall, these evolutionarily conserved CTL epitopes should be useful as part of the immunogen s in an HIV 1 vaccine for prophylaxis and post infection therapy.
87 LIST O F REFERENCES  Barre Sinoussi F, Cherman JC, Rey R, et al. Isolation of a T lymphotrophic retrovirus from a patient at risk for acquired immune deficiency syndrome (AIDS). Science 1983; 220: 868 1.  Cohen MS, Hellmann N, Levy JA, DeCock K, Lan ge J. The spread, treatment, and prevention of HIV 1: evolution of a global pandemic. J Clin Invest 2008; 118: 1244 4. [3 ] UNAIDS 2010 AIDS Epidemic Update (November 2010). Accesed December 12, 2011. [http://www.unaids.org/en/knowledgecentre/hivdata/e pidemiology/epislides.asp].  Plotkin SA. Vaccines: correlates of vaccine induced immunity. Clin Infect Dis 2008; 47: 401 9.  Robinson HL, Amara RR. T cell vaccines for microbial infections. Nat Med 2005; 11: 4.  Berkhout B, Verhoef K. Evolution of live attenuated virus vaccines. Dev Biol (Basel) 2001; 106: 217 1.  Brown, F. 1993. Review of accidents caused by incomplete inactivation of viruses. Dev Biol Stand 1993; 81: 103 7.  Taylor BS, Sobieszczyk ME, McCutchan FE, Hamme r SM. The challenge of HIV 1 subtype diversity. N Engl J Med 2008; 358: 1590 602.  McBurney SP, Ross TM. Viral sequence diversity: Challenges for AIDS vaccine designs. Expert Rev Vaccines 2008; 7: 1405 7.  Walker LM, Phogat SK, Chan Hui PY,Wag ner D, Phung P, et al. Broad and potent neutralizing antibodies from an African donor reveal a new HIV 1 vaccine target. Science 2009; 326: 285 9.  Burton DR, Desrosiers RC, Doms RW, Koff WC, Kwong PD, Moore JP, et al. HIV vaccine design and the neut ralizing antibody problem. Nat Immunol 2004; 5: 233 6.  Gamble LJ, Matthews QL. Current progress in the development of a prophylactic vaccine for HIV 1. Drug Des Devel Ther 2010; 5: 9 6.  Levy JA. HIV and the pathogenesis of AIDS. 3rd ed. Washi ngton, DC: ASM Press 2007.
88  Warren J. Preclinical AIDS vaccine research: survey of SIV, SHIV, and HIV challenge studies in vaccinated nonhuman primates. J Med Primatol 2002; 4 5: 237 6.  Joag SV. Primate models of AIDS. Microbes Infect 2000; 2: 223 9.  Igarashi T, Imamichi H, Brown CR, Hirsch VM, Martin MA. The emergence and characterization of macrophage tropic SIV/HIV chimeric viruses (SHIVs) present in CD4 T cell depleted rhesus monkeys. J Leukoc Biol 2003; 74: 772 80.  Leroux C, Cadore JL, Montelaro RC. Equine Infectious Anemia Virus (EIAV): what has HIV's country cousin got to tell us? Vet Res 2004; 35: 485 512.  Narayan O, Joag SV, Stephens EB. Selected models of HIV induced neurological disease. Curr Top Microbiol Immun ol 1995; 202: 151 66.  Zink MC, Narayan O, Kennedy PG, Clements JE. Pathogenesis of visna/maedi and caprine arthritis encephalitis: new leads on the mechanism of restricted virus replication and persistent inflammation. Vet Immunol Immunopathol 1987; 15: 167 80.  Yamamoto JK, Pu R, Sato E, Hohdatsu T. Feline immunodeficiency virus pathogenesis and development of a dual subtype feline immunodeficiency virus vaccine. AIDS 2007; 21: 547 63.  Yamamoto JK, Sanou MP, Abbott JR, Coleman JK. Feli ne immunodeficiency virus model for designing HIV/AIDS vaccines. Curr HIV Res 2010; 8: 14 25.  Maanen M, Sutton RE. Rodent models for HIV 1 infection and disease. Cur HIV Res 2003; 1: 121 30.  Berges BK, Rowan MR. The utility of the new generat ion of humanized mice to study HIV 1 infection: transmission, prevention, pathogenesis, and treatment. Retrovirology 2011; 8: 65.  Watanabe S, Ohta S, Yajima M. Humanized NOD/SCID/IL2Rgammanull mice transplanted with hematopoietic stem cells under no nmyeloablative conditions show prolonged life spans and allow detailed analysis of human immunodeficiency virus type 1 pathogenesis. J Virol 2007; 81: 13259 64.  Watanabe S, Terashima K, Ohta S Hematopoietic stem cell engrafted lasting HIV 1 infection with specific humoral immune responses. Blood 2007; 109: 212 8.  Flynn NM, Forthal DN, Harro CD, et al. Placebo control led phase 3 trial of a recombinant glycoprotein 120 vaccine to prevent HIV 1 infection. J Infect Dis 2005; 191: 654 65.
89  Pitisuttithum P, Nitayaphan S, Thongcharoen P, et al. Safety and immunogenicity of combinations of recombinant subtype E and B h uman immunodeficiency virus type 1 envelope glycoprotein 120 vaccines in healthy Thai adults. J Infect Dis 2203; 188: 219 27.  Pitisuttithum P, Gilbert P, Gurwith M, et al. Randomized, double blind, placebo controlled efficacy trial of a bivalent re combinant glycoprotein 120 HIV 1 vaccine among injection drug users in Bangkok, Thailand. J Infect Dis 2006; 194: 1661 71.  Buchbinder SP, Mehrotra DV, Duerr A, et al. Efficacy assessment of a cell mediated immunity HIV 1 vaccine (the Step Study): a double blind, randomised, placebo controlled, test of concept trial. Lancet 2008; 372: 1881 93.  McElrath MJ, De Rosa SC, Moodie Z, et al. HIV 1 vaccine induced immunity in the test of concept Step Study: a case cohort analysis. Lancet 2008; 372: 1 894 905.  Auvert B, Taljaard D, Lagarde E, Sobngwi Tambekou J, Sitta R, Puren A. Randomized, controlled intervention trial of male circumcision for reduction of HIV infection risk: the ANRS 1265 trial. PLoS Med 2005; 2(11): e298.  Duerr A, Hu ang Y, Buchbinder S, et al Extended Follow up Confirms Early Vaccine Enhanced Risk of HIV Acquisition and Demonstrates Waning Effect Over Time Among Participants in a Randomized Trial of Recombinant Adenovirus HIV Vaccine (Step Study). J Infect Dis 2012; 206: 258 66.  Curlin ME, Cassis Ghavami F, Magaret AS, et al Serological immunity to adenovirus serotype 5 is not associated with risk of HIV infection: a case control study. AIDS 2011; 25: 153 8.  Rerks Ngarm S, Pitisuttithum P, Nitayapha n S, et al. MOPH TAVEG Investigators. Vaccination with ALVAC and AIDSVAX to prevent HIV 1 infection in Thailand. N Engl J Med 2009; 361: 2209 20.  Nitayaphan S, Pitisuttithum P, Karnasuta C, et al. Safety and immunogenicity of an HIV subtype B and E prime boost vaccine combination in HIV negative Thai adults. J Infect Dis 2004; 90: 702 6.  Karnasuta C, Paris RM, Cox JH, et al. Antibody dependent cell mediated cytotoxic responses in participants enrolled in a phase I/II ALVAC HIV/AIDSVAX B/E prime boost HIV 1 vaccine trial in Thailand. Vaccine 2005; 23: 2522 9.  Baden LR, Dolin R. Th e road to an effective HIV vaccine N Engl J Med 2012; 366:1343 4.
90  Callaway E. Clues emerge to explain first successful HIV vaccine trial. Nature 2011; doi:10.1038/news.2011.541. Accessed January 22 2012. http://www.nature.com/news/2011/110916/full /news.2011.541.html  The International AIDS Vaccine Initiative. Database of AIDS Vaccine Candidates in Clinical Trials: http://www.iavireport.org/trials db/Pages/default.aspx.  Koup RA, Graham BS, Douek DC. The quest for a T cell based immun e correlate of protection against HIV: a story of trials and errors. Nat Rev Immunol 2011; 11:65 70.  Rolland M, Gilbert P. Evaluating Immune Correlates in HIV Type 1 Vaccine Efficacy Trials: What RV144 May Provide. AIDS Res Hum Ret 2011; DOI: 10.10 89/aid.2011.0240.  Young JM, Turpin JA, Musib R, Sharma OK. Outcomes of a National Institute of Allergy and Infectious Diseases Workshop on Understanding HIV Exposed but Seronegative Individuals. AIDS Res Hum Retroviruses 2011; 27: 737 43.  T omescu C, Abdulhaqq S, Montaneb LJ. Evidence for the innate immune response as a correlate of protection in human immunodeficiency virus (HIV) 1 highly exposed seronegative subjects (HESN). Clin Exp Immunol 2011; 164: 158 69.  Deeks SG, Walker BD. H uman immunodeficiency virus controllers: mechanisms of durable virus control in the absence of antiretroviral therapy. Immunity 2007; 27: 406 16.  Hubert JB, Burgard M, Dussaix E, et al. Natural history of serum HIV 1 RNA levels in 330 patients with a known date of infection. The SEROCO Study Group. AIDS 2000; 14: 123 31.  Sedaghat AR, Rastegar DA, O'Connell KA, Dinoso JB, Wilke CO, Blankson JN. T cell dynamics and the response to HAART in a cohort of HIV 1 infected elite suppressors. Clin Inf ect Dis 2009; 49:1763 6.  Sajadi MM, Constantine NT, Mann DL, et al Epidemiologic characteristics and natural history of HIV 1 natural viral controllers, J Acquir Immune Defic Syndr 2009; 50:403 08.  Blankson JN. Effector mechanisms in HIV 1 infected elite controllers: highly active immune responses? Antiviral Res 2010; 85: 295 302.  Barker E, Mackewicz CE, Reyes Teran G et al. Virological and immunological features of long term human immunodeficiency virus infected individuals who h ave remained asymptomatic compared to those who have progressed to acquired immunodeficiency syndrome. Blood 1998 92: 3105 14.
91  Saez Cirion A, Lacabaratz C, Lambotte O, et al. HIV controllers exhibit potent CD8 T cell capacity to suppress HIV infect ion ex vivo and peculiar cytotoxic T lymphocyte activation phenotype. Proc Natl Acad Sci 2007; 104: 6776 81.  Phillips RE, Rowland Jones S, Nixon DF, et al. Human immunodeficiency virus genetic variation that can escape cytotoxic T cell recognition. Nature 1991; 354: 453 9.  O'Brien SJ, Nelson GW. Human genes that limit AIDS. Nat Genet 2004; 36: 565 74.  Gao X, Nelson GW, Karacki P, et al. Effect of a single amino acid change in MHC class I molecules on the rate of progression to AIDS. N Engl J Med 2001; 344: 1668 75.  Pereyra F, Jia X, McLaren JP, et al. The major genetic determinants of HIV 1 Science 2010; 330: 1551 7.  McMichael AJ, Jones EY. Genetics. First class contro l of HIV 1. Science 2010; 330: 1488 90.  Killian MS, Levy JA. HIV/AIDS: 30 years of progress and future challenges. Eur J Immunol 2011; 41: 3401 11.  Walker CM, Moody DJ, Stites DP, Levy JA. CD8 lymphocytes can control HIV infection in vitro by suppressing virus replication. Science 1986; 234: 1563 6.  Walker BD, Flexner C, Paradis TJ, et al HIV 1 reverse transcriptase is a target for cytotoxic T lymphocytes in infected individuals. Science 1988; 240: 64 6.  Koup RA, Safrit JT, Cao Y, et al. Temporal association of cellular immune responses with the initial control of viremia in primary human immunodeficiency virus type 1 syndrome. J Virol 1994; 68: 4650 5.  Cao Y, Qin L, Zhang L, Safrit J, Ho DD. Virologic and immunologic characterization of long term survivors of human immunodeficiency virus type 1 infection. N Engl J Med 1995; 332: 201 8.  Betts MR, Krowka JF, Kepler TB, et al. Human immunodeficiency virus type 1 specific cytotoxic T lymphocyte activity is inversel y correlated with HIV type 1 viral load in HIV type 1 infected long term survivors. AIDS Res Hum Retroviruses 1999; 15: 1219 28.  Brown DM. Cytolytic CD4 cells: Direct mediators in infectious disease and malignancy. Cell Immunol 2010; 262: 89 95. [6 3] Appay VJ, Zaunders J, Papagno L, et al. Characterization of CD4 CTLs ex vivo. J Immunol 2002: 168; 5954 8.
92  Soghoian DM, Jessen H, Flanders M et al HIV Specific Cytolytic CD4 T Cell Responses During Acute HIV Infection Predict Disease Outco me. Sci Transl Med 2012; 123:1123 5.  Horowitz A, Behrens RH, Okell L, Fooks AR, Riley EM. NK cells as effectors of acquired immune responses: effector CD4 T cell dependent activation of NK cells following vaccination. J Immunol 2010; 185: 2808 18.  Sun JC, Beilke JN, Lanier LL. Adaptive immune features of natural killer cells. Nature 2009; 457: 557 61.  Sun JC, Lanier LL. Natural killer cells remember: an evolutionary bridge between innate and adaptive immunity? Eur J Immunol 2009; 39 : 2059 64.  Berger CT, Alter G. Natural killer cells in spontaneous control of HIV infection. Curr Opin HIV AIDS 2011; 6: 208 13.  Tiemessen CT, Shalekoff S, Meddows Taylor S, et al. Cutting Edge: Unusual NK cell responses to HIV 1 peptides a re associated with protection against maternal infant transmission of HIV 1. J Immunol 2009; 182: 5914 8.  Montoya CJ, Velilla PA, Chougnet C, Landay AL, Rugeles MT. Increased IFN gamma production by NK and CD3 + /CD56 + cells in sexually HIV 1 exposed but uninfected individuals. Clin Immunol 2006; 120 : 138 46.  Tiemessen CT, Shalekoff S, Meddows Taylor S, et al. Natural killer cells that respond to human immunodeficiency virus type 1 (HIV 1) peptides are associated with control of HIV 1 infectio n. J Infect Dis 2010; 202: 1444 53.  Shibata R, Igarashi T, Haigwood N, et al Neutralizing antibody directed against the HIV 1 envelope glycoprotein can completely block HIV 1/SIV chimeric virus infections of macaque monkeys. Nat Med 1999; 5: 204 10.  Trkola A, Kuster H, Rusert P, et al. Delay of HIV 1 rebound after cessation of antiretroviral therapy through passive transfer of human neutralizing antibodies. Nat Med 2005; 11: 615 22.  Bailey JR, Lassen KG, Yang HC, et al. Neutralizin g antibodies do not mediate suppression of human immunodeficiency virus type 1 in elite suppressors or selection of plasma virus variants in patients on highly active antiretroviral therapy. J Virol 2006; 80: 4758 70.  Harrer T, Harrer E, Kalams SA, et al. Strong cytotoxic T cell and weak neutralizing antibody responses in a subset of persons with stable nonprogressing HIV type 1 infection. AIDS Res Hum Retroviruses 1996; 12: 585 92.
93  Pereyra F, Addo MM, Kaufmann DE, et al. Genetic and immunol ogic heterogeneity among persons who control HIV infection in the absence of therapy. J Infect Dis 2008; 197: 563 71.  Lambotte O, Ferrari G, Moog C, et al. Heterogeneous neutralizing antibody and antibody dependent cell cytotoxicity responses in HI V 1 elite controllers. AIDS 2009; 23: 897 906.  Emu B, Sinclair E, Hatano H, et al. HLA class I restricted T cell responses may contribute to the control of human immunodeficiency virus infection, but such responses are not always necessary for long term virus control. J Virol 2008; 82: 5398 407.  Plotkin, SA. Immunologic correlates of protection induced by vaccination. Pediatr Infect Dis 2001; 20 : 73 5  Karlsson Hedestam GB, Fouchier RA, Phogat S, Burton DR, Sodroski J, Wyatt RT. The c hallenges of eliciting neutralizing antibodies to HIV 1 and to influenza virus. Nat Rev Microbiol 2008; 6: 143 55.  Sette A, Sidney J. HLA supertypes and supermotifs: a functional perspective on HLA polymorphism. Curr Opin Immunol 1998; 4: 478 82. [8 2] Sidney J, Peters B, Frahm N, Brander C, Sette A. HLA class I supertypes: a revised and updated classification. 2008; BMC Immunol: 9 :1.  Greenbaum J, Sidney J, Chung J, Brander C, Peters B, Sette A. Functional classification of class II human l eukocyte antigen (HLA) molecules reveals seven different supertypes and a surprising degree of repertoire sharing across supertypes. Immunogenetics 2011; 63: 325 35.  Leslie A, Price DA Mkhize P, et al Differential selection pressure exerted on H IV by CTL targeting identical epitopes but restricted by distinct HLA alleles from the same HLA supertype. J. Immunol. 2006; 177 : 4699 708.  Hladik F, McElrath MJ. Setting the stage: host invasion by HIV. Nat Rev Immunol 2008; 8: 447 57.  F rankel SS, Wenig BM, Burke AP, et al. Replication of HIV 1 in dendritic cell derived syncytia at the mucosal surface of the adenoid. Science 1996; 272: 115 7.  Poles MA, Boscardin WJ, Elliott J, et al. Lack of decay of HIV 1 in gut associated lympho id tissue reservoirs in maximally suppressed individuals. J Acquir Immune Defic Syndr 2006; 43: 65 8.  Lefrancois L, Puddington L. Intestinal and pulmonary mucosal T cells: local heroes fight to maintain the status quo. Annu Rev Immunol 2006; 24: 68 1 704.
94  Ibarrondo FJ, Anton PA, Fuerst M, et al. Parallel human immunodeficiency virus type 1 specific CD8 T lymphocyte responses in blood and mucosa during chronic infection. J Virol 2005; 79: 4289 97.  Musey L, Ding Y, Cao J, et al. Ontogeny and specificities of mucosal and blood human immunodeficiency virus type 1 specific CD8 (+) cytotoxic T lympho cytes. J Virol 2003; 77: 291 300.  Ferre AL, Lemongello D, Hunt PW, et al. Immunodominant HIV specific CD8 T cell responses are common to blood and gastrointestinal mucosa, and Gag specific responses dominate in rectal mucosa of HIV controllers. J Virol 2010; 84: 10354 65.  Shacklett BL, Cu Uvin S, Beadle TJ, et al. Quantification of HIV 1 specific T cell responses at the mucosal cervicovaginal surface. AIDS 2000; 14: 1911 5.  Genesca M Characterization of an Effective CTL Response ag ainst HIV and SIV Infections. 2011; 2011: 103924.  Schultheiss T, Reiner Schulte R, Ulrike Sauermann U, Wiebke Ibing W, Stahl Hennig C. Strong mucosal immune responses in SIV infected macaques contribute to viral control and preserved CD4 T cell levels in blood and mucosal tissues. Retrovirol 2011; 8: 24.  Murphy Corb M, Wilson LA, Trichel AM, et al. Selective induction of protective MHC class I restricted CTL in the intestinal lamina propria of rhesus monkeys by transient SIV infe ction of the colonic mucosa. J Immunol 1999; 162: 540 9.  Fuller DH, Rajakumar P, Che JW et al Therapeutic DNA Vacci ne Induces Broad T Cell Responses in the Gut and Sustained Protection from Viral Rebound and AIDS in SIV Infected Rhesus Macaques. PLoS ONE 2012; 7: e33715.  Edwards BH, Bansal A, Sabbaj S, Bakari J, Mulligan MJ, Geoepfert PA. Magnitude of function al CD8 T cell responses to the Gag protein of human immunodeficiency virus type 1 correlates inversely with viral load in plasma. J Virol 2002; 76: 2298 305.  Rosenberg ES, Billingsley JM, Caliendo AM, et al Vigorous HIV 1 specific CD4 T cell res ponses associated with control of viremia. Science 1997; 278 : 1447 50.  Simons BC, Vancompernolle SE, Smith RM, et al Despite biased TRBV gene usage against a dominant HLA B57 restricted epitope, TCR diversity can provide recognition of circulating epitope variants. J Immunol 2008; 181 : 5137 46.  Betts MR, Nason MC, West SM, et al. HIV nonprogressors preferentially maintain highly functional HIV specific CD8 T cells. Blood 2006; 107 : 4781 9.
95  Day CL, Kiepiela P, Leslie AJ, et al Proliferative capacity of epitope specific CD8 T cell responses is inversely related to viral load in chronic human immunodeficiency virus type 1 infection J Virol 2007; 81 : 434 8.  Migueles SA, Osborne CM, Royce C, et al. Lytic granule loading of CD8 T cells is required for HIV infected cell elimination associated with immune control. Immunity 2008 ; 29 : 1009 21.  Blackbourn DJ, Mackewicz CE, Baker E, et al Suppr ession of HIV replication by lymphoid tissue CD8 cells correlates with the clinical state of HIV infected individuals. Proc Natl Acad Sci 1996; 93 : 13125 30.  Addo MM, Draenert R, Rathod A, et al. Fully differentiated HIV 1 specific CD8 T effector cells are more frequently detectable in controlled than in progressive HIV 1 infection. PLoS ONE 2007; 2, e321.  Burgers WA, Riou C, Mlotshwa M, et al. Association of HIV specific and total CD8 T memory phenotypes in subtype C HIV 1 infection with viral set point. J Immunol 2009; 182: 4751 61.  Day CL, Kaufmann DE, Kiepiela P, et al PD 1 expression on HIV specific T cells is associated with T cell exhaustion and disease progression. Nature 2006; 443 : 350 4.  Kiepiela P, Kholiswa N, Thob akgale C, et al CD8 T cell responses to different HIV proteins have discordant associations with viral load. Nature Med 2007; 13 : 46 53.  Zuniga R, Lucchetti A, Galvan P, et al Relative dominance of Gag p24 specific cytotoxic T lymphocytes is associated with human im munodeficiency virus control. J Virol 2006; 80: 3122 25.  Migueles SA, Sabbaghian MS, Shupert WL, et al HLA B*5701 is highly associated with restriction of virus replication in a subgroup of HIV infected long term nonprogressors. Proc Natl Acad Sci USA 2000; 97 : 2709 14.  Turnbull EL, Lopes AR, Jones NA, et al HIV 1 epitope specific CD8 T cell responses strongly associated with delayed disease progression cross recognize epitope variants efficiently. J Immunol 2006; 176 : 6130 46.  Schneidewind A, Brockman MA, Yang R, et al Escape from the dominant HLA B27 restricted cytotoxic T l ymphocyte response in Gag is associated with a dramatic reduction in human immunodeficiency virus type 1 replication. J Virol 2007; 81: 12382 93.  Jin HT, Anderson AC, Tan WG, et al Cooperation of Tim 3 and PD 1 in CD8 T cell exhaustion during chron ic viral infection. Proc Natl Acad Sci 2010; 107: 14733 8.
96  Khaitan A Unutmaz D. Revisiting immune exhaustion during HIV infection. Curr HIV/AIDS Rep 2011; 8 : 4 11.  Zhang JY, Zhang Z,Wang X, et al. PD 1 up regulation is correlated with HIV sp ecific memory CD8 T cell exhaustion in typical progressors but not in long term nonprogressors. Blood 2007; 109: 4671 8.  Frahm N, DeCamp AC, Friedrich DP, et al Human adenovirus specific T cells modulate HIV specific T cell responses to an Ad5 vec tored HIV 1 vaccine. J Clin Invest 2012; 122: 359 67.  Li F, Finnefrock AC, Dubey SA, et al Mapping HIV 1 vaccine induced T cell responses: bias towards less conserved regions and potential impact on vaccine efficacy in the Step study. PLoS One 201 1; 6:e20479.  Fitzgerald DW, Janes H, Robertson M, et al An Ad5 vectored HIV 1 vaccine elicits cell mediated immunity but does not affect disease progression in HIV 1 infected male subjects: results from a randomized placebo controlled trial (the S tep study). J Infect Dis 2011; 203: 765 72.  Gray G, Buchbinder S, Duerr A. Overview of STEP and Phambili trial results: two phase IIb test of concept studies investigating the efficacy of MRK adenovirus type 5 gag/pol/nef subtype B HIV vaccine. Cur r Opin HIV AIDS 2010; 5:357 61.  Barouch DH, Liu J, Li H, et al Vaccine protection against acquisition of neutralization resistant SIV challenges in rhesus monkeys. Nature 2012; 482: 89 93.  Hansen SG, Ford JC, Lewis MS, et al Profound earl y control of highly pathogenic SIV by an effector memory T cell vaccine. Nature 2011; 473: 523 7.  de Souza MS, Ratto Kim S, Chuenarom W, et al The Thai phase III trial (RV144) vaccine regimen induces T cell responses that preferentially target epi topes within the V2 region of HIV 1 envelope. J Immunol 2012; 188:5166 76.  Rolland M, Nickle DC, Mullins JI. HIV 1 group M conserved elements vaccine. PLoS Pathog 2007; 3: e157.  De Groot AS, Marcon L, Bishop EA, et al. HIV vaccine developmen t by computer assisted design: The GAIA vaccine. Vaccine 2005; 23: 2136 48.  of comprising linked minigenes, confers protection from lethal dose virus challenge. J Virol 1993; 67: 348 52.  Moise L, Buller RM, Schriewer J, et al. VennVax, a DNA prime, peptide boost multi T cell epitope poxvirus vaccine, induces protective immunity against vaccinia infection by T cell response alone. Vaccine 2011; 29: 501 11.
97  Moise L, Ar dito M, Desrosiers J, et al Immunome derivedEpitope drivenVaccines (ID EDV) Protect against Viral or Bacterial Challenge in Humanized Mice. Procedia Vaccinol 2009; 1: 15 22.  Hanke T, McMichael AJ, Mwau M, et al. Development of a DNA MVA/HIVA vaccin e for Kenya. Vaccine 2002; 20: 1995 8  Hanke T, McMichael A J. Design and construction of an experimental HIV 1 vaccine for a year 2000 clinical trial in Kenya. Nat Med 200; 6: 951 5.  Mwau MI, Cebere J, Sutton P, et al. A human immunodeficien cy virus 1 (HIV 1) clade A vaccine in clinical trials: stimulation of HIV specific T cell responses by DNA and recombinant modified vaccinia virus Ankara (MVA) vaccines in humans. J Gen Virol 2004; 85: 911 9.  Cebere I, Dorrell L, McShane H, et al P hase I clinical trial safety of DNA and modified virus Ankara vectored human immunodeficiency virus type 1 (HIV 1) vaccines administered alone and in a prime boost regime to healthy HIV 1 uninfected volunteers. Vaccine 2006; 24: 417 25.  Jaoko W, N akwagala FN, Anzala O, et al. Safety and immunogenicity of recombinant low dosage HIV 1 A vaccine candidates vectored by plasmid pTHr DNA or modified vaccinia virus Ankara (MVA) in humans in East Africa. Vaccine 2008; 26: 2788 95.  Hanke T, McMichael AJ, Dorrell L. Clinical experience with plasmid DNA and modified vaccinia virus Ankara vectored human immunodeficiency virus type 1 clade A vaccine focusing on T cell induction. J Gen Virol 2007; 88: 1 12.  Goonetilleke N, Moore S, Dally L, et al Induction of multifunctional human immunodeficiency virus type 1 (HIV 1) specific T cells capable of proliferation in healthy subjects by using a prime boost regimen of DNA and modified vaccinia virus Ankara vectored vaccines expressing HIV 1 Gag coupled to CD8 T cell epitopes. J Virol 2006; 80: 4717 28.  Gorse GJ, Baden LR, Wecker M, et al. Safety and immunogenicity of cytotoxic T lymphocyte poly epitope, DNA plasmid (EP HIV 1090) vaccine in healthy, human immunodeficiency virus type 1 (HIV 1) uninfected adults. Vaccine 2008; 26: 215 23.  Spearman P, Kalams S, Elizaga M, Metch B, Chiu YL, et al Safety and immunogenicity of a CTL multiepitope peptide vaccine for HIV with or without GM CSF in a phase I trial. Vaccine 2009; 27: 243 9.  Salmon Ce ron D, Durier C, Desaint C. et al Immunogenicity and safety of an HIV 1 lipopeptide vaccine in healthy adults: a phase 2 placebo controlled ANRS trial. AIDS 2010; 24: 2211 23.
98  Abbas AK Lichtman AH, Pillai S. Cellular and Molecular Immunology, Phi ladelphia: Elsevier, 6th edition, 2007.  Keating SM, Bejon P, Berthoud T, et al Durable human memory T cells quantifiable by cultured ELISPOT assays are induced by heterologous prime boost immunization and correlate with protection against malaria. J Immunol 2005; 175: 5675 80.  Gahery H, Daniel N, Charmeteau B, et al New CD4 and CD8 T cell responses induced in chronically HIV type 1 infected patients after immunizations with an HIV type 1 lipopeptide vaccine. AIDS Res Hum Retroviruses 2006; 2 2: 684 94.  Jin X, Newman MJ, De Rosa S, et al A novel HIV T helper epitope based vaccine elicits cytokine secreting HIV specific CD4 T cells in a Phase I clinical trial in HIV uninfected adults. Vaccine 2009; 27: 7080 6.  Dorrell L, Yang H, O ndondo B, et al Expansion and diversification of virus specific T cells following immunization of human immunodeficiency virus type 1 (HIV 1) infected individuals with a recombinant modified vaccinia virus Ankara/HIV 1 Gag vaccine. J Virol 2006; 80: 4705 16.  Ondondo BO, Yang H, Dong T, et al Immunisation with recombinant modified vaccinia virus Ankara expressing HIV 1 gag in HIV 1 infected subjects stimulates broad functional CD4 T cell responses. Eur J Immunol 2006; 36: 2585 94.  Slyker JA Lohman BL, Mbori Ngacha DA, et al Modified vaccinia Ankara expressing HIVA antigen stimulates HIV 1 specific CD8 T cells in ELISpot assays of HIV 1 exposed infants. Vaccine 2005; 23: 4711 9.  Berzofsky J, Ahlers J, Belyakov I. Strategies for desig ning and optimizing new generation vaccines. Nature Rev Immunol 2001; 1: 209 19.  Ahlers JD, Dunlop N, Alling DW, Nara PL, Berzofsky JA. Cytokine in adjuvant steering of the immune response phenotype to HIV 1 vaccine constructs: GM CSF and TNF syner gize with IL 12 to enhance induction of CTL. J Immunol 1997; 158: 3947 58.  Disis ML, Bernhard H, Shiota FM, et al Granulocyte macrophage colony stimulating factor: an effective adjuvant for protein and peptide based vaccines. Blood 1996; 88: 202 10  Belyakov IM, Ahlers JD, Clements JD, Strober W, Berzofsky JA. Interplay of cytokines and adjuvants in the regulation of mucosal and systemic HIV specific cytotoxic T lymphocytes. J Immunol 2000; 165: 6454 62.  Klinman DM., Yi A K, Beaucage S L, Conover J, Krieg AM. CpG motifs present in bacterial DNA rapidly induce lymphocytes to secrete interleukin 6, interleukin 12, and interferon gamma. Proc Natl Acad Sci 1996; 93 : 2879 83.
99  Evans TG, Keefer MC, Weinhold KJ, et al A canarypox vaccine expressing multiple human immunodeficiency virus type 1 genes given alone or with rgp120 elicits broad and durable CD8 cytotoxic T lymphocyte responses in seronegative volunteers. J Infect Dis 1999; 180 : 290 8.  Lubeck MD, Natuk RJ, Chengalvala M, et al Immunogenicity of recombinant adenovirus human immunodeficiency virus vaccines in chimpanzees following intranasa l administration. AIDS Res Hum Retroviruses 1994; 10: 1443 9.  Letourneau S, Im EJ, Mashishi T, et al Design and pre clinical evaluation of a universal HIV 1 vaccine. PLoS ONE 2007; 2: e984.  Li F, Horton H, Gilbert PB, McElrath JM, Corey L, Se lf SG. HIV 1 CTL based vaccine immunogen selection: antigen diversity and cellular response features. Curr HIV Res 2007; 5: 97 107.  Korber BT, Letvin NL, Haynes BF. T cell vaccine strategies for human immunodeficiency virus, the virus with a thousan d faces. J Virol 2009; 83: 8300 14.  Cao H, Kanki P, Sankale JL, et al. Cytotoxic T lymphocyte cross reactivity among different human immunodeficiency virus type 1 clades: implications for vaccine development. J Virol 1997; 71: 8615 23.  Rowlan d Jones SL, Dong T, Fowke KR, et al. Cytotoxic T cell responses to multiple conserved HIV epitopes in HIV resistant prostitutes in Nairobi. J Clin Invest 1998; 102: 1758 65.  Johnson RP, Trocha A, Yang L, et al. HIV 1 gag specific cytotoxic T lymphoc ytes recognize multiple highly conserved epitopes. Fine specificity of the gag specific response defined by using unstimulated peripheral blood mononuclear cells and cloned effector cells. J Immunol 1991; 147: 1512 21.  Smith SM. HIV CTL escape: at w hat cost? Retrovirology 2004; 1: 8.  Wang YE, Li B, Carlson JM. Protective HLA class I alleles that restrict acute phase CD8 T cell responses are associated with viral escape mutations located in highly conserved regions of human immunodeficiency virus type 1. J Virol 2009; 83: 1845 55.  Santra S, Liao HX, Zhang R, et al. Mosaic vaccines elicit CD8 T lymphocyte response s that confer enhanced immune coverage of diverse HIV strains in monkeys. Nat Med 2010; 16: 324 8.  et al. Mosaic HIV 1 vaccines expand the breadth and depth of cellular immune responses in rhesus monkeys. Nat Med 2010; 16: 319 23.
100  Yusim K, Kesmir C, Gaschen B, et al. Clustering patterns of cytotoxic T lymphocyte epitopes in human immunodeficiency virus type 1 (HIV 1) proteins reveal imprints of immune evasion on HIV 1 global variation. J Virol 2002; 76: 875 7 68.  Jenner E. An inquiry into the causes and effects of the Variolae Vaccinae, a disease discovered in some of the western counties of England, particularly Gloucestershire, and known by the name of the cow pox. London: Sampson Low, 1798.  Ardito M Fueyo J Tassone R et al An integrated genomic and immunoinformatic approach to H. pylori vaccine design. Immunome Res 2011; 20; 7: 1.  Moss SF, Moise L, Lee DS, et al HelicoVax: epitope based therapeutic Helicobacter pylori vaccination in a mou se model. Vaccine 2011; 29: 2085 91.  De Groot AS, Rivera DS, McMurry JA, Buus S, Martin W. Identification of immunogenic HLA HIV. Vaccine 2008; 26: 3059 71.  Llano A, Frahm N, Bra nder C: How to optimally define optimal cytotoxic T lymphocyte epitopes in HIV infection? In HIV Molecular Immunology. Edited by: Yusim K, Korber BTM, Brander C, et al. Los Alamos National Laboratory; 2009:3 24.  Yongqun H, Rappuoli R, De Groot AS, C hen RT. Emerging Vaccine Informatics. J Biomed Biotechnol 2010; 2010: 218590.  Bhasin M, Raghava, GPS. Prediction of CTL epitopes using QM, SVM and ANN techniques. Vaccine 2004; 22: 3195 201.  Larsen MV, Lundegaard C, Lamberth K, Buus S, Lund O Nielsen M. Large scale validation of methods for cytotoxic T lymphocyte epitope prediction. BMC Bioinform 2007; 8: 424.  Stranzl T, Larsen MV, Lundegaard C, Nielsen M. NetCTLpan: pan specific MHC class I pathway epitope predictions. Immunogenetics 2010; 62: 357 68.  Lundegaard C, Lamberth K, Harndahl M, Buus S, Lund O, Nielsen M. NetMHC 3.0: Accurate web accessible predictions of Human, Mouse, and Monkey MHC class I affinities for peptides of length 8 11. NAR 2008; 36 : 50912.
101  Abbott JR, Sanou MP, Coleman JK, Yamamoto JK. Evolutionarily conserved T cell epitopes on FIV for designing an HIV/AIDS vaccine. Vet Immunol and Immunopathol 2011; 143: 246 54.  Lichterfeld M, Kaufmann DE, Yu XG, et al Loss of HIV 1 specific CD8+ T cell p roliferation after acute HIV 1 infection and restoration by vaccine induced HIV 1 specific CD4+ T cells. J Exp Med 2004; 200: 701 12.  Lichterfeld M, Kaufmann DE, Yu XG, et al Loss of HIV 1 specific CD8+ T cell proliferation after acute HIV 1 infect ion and restoration by vaccine induced HIV 1 specific CD4+ T cells. J Exp Med 2004; 200: 701 12. [ 175 ] Padian NS, Isbell MT, Russell ES, Essex M. The Future of HIV Prevention. J Acquir Immune Defic Syndr 2012; 60: S22 6. [ 17 6 ] Mothe B, Liano A, Ibarrondo J, et al Definition of the viral targets of protective HIV 1 specific T cell responses. J Transl Med 2011; 9 : 208. [ 177 ] Zolla Pazner S. Identifying epitopes of HIV 1 that induce protective antibodies. Nat Rev Immunol 2004; 4: 199 210. [ 178 ] Stamatatos L. HIV vaccine design: the neutralizing antibody conundrum. Curr Opin Immunol 2012; 24: 316 23. [ 179 ] Schmitz JE, Kuroda MJ, Santra S, et al Control of viremia in simian immunodeficiency virus infection by CD8+ lymphocytes. Science 1999; 283: 857 60. [ 180 ] Robinson WE, Kawamura T, Gorny MK, et al Human monoclonal antibodies to the human immunodeficiency virus type 1 (HIV 1) transmembrane glycoprotein gp41 enhance HIV 1 infection in vitro. Proc Natl Acad Sci USA 1990; 87: 3185 89.  Leslie AJ, Pfafferott KJ, Chetty P, et al HIV evolution: CTL escape mutation and reversion after transmission. Nat Med 2004; 10: 282 9. [ 182 ] Troyer RM, McNevin J, Liu Y, et al Variable fitness impact of HIV 1 escape mutations to cytotoxic T lymphocyte (CTL) response. PLoS Pathog 2009; 5:e1000365. [ 183 ] Goulder PJ,Watkins DI. Impact of MHC class I diversity on immune control of immunodeficiency virus replication. Nat Rev Immunol 2008; 8: 619 30. [ 18 4 ] Balla Jhagjhoorsingh SS, Koopman G, Mooij P, et al Conserved CTL epitopes shared between HIV infected human long term survivors and chimpanzees. J Immunol 1999; 162: 2308 14.
102 [ 185 ] Walther Jallow L, Nilsson C, Soderlund J, et al Cross protection against mucosal simian immunodeficiency virus (SIVsm) chal lenge in human immunodeficiency virus type 2 vaccinated cynomolgus monkeys. J. Gen Virol 2001; 82: 1601 12. [ 186 ] Abbott JR, Pu R, Coleman JK, Yamamoto JK. Utilization of feline ELISPOT for mapping vaccine epitopes. Methods Mol Biol 2012; 792: 47 63. [ 18 7 ] Horton H, Thomas EP, Stucky JA, et al Optimization and validation of an 8 color intracellular cytokine staining (ICS) assay to quantify antigen specific T cells induced by vaccination J Immunol 2007; 323: 39 54. [ 188 ] McKinnon LR, Kaul R, Kimani J, et al HIV specific CD8+ T cell proliferation is prospectively associated with delayed disease progression. Immunol Cell Biol 2012; 90: 346 51. [ 189 ] Bouhdoud L, Villain P, Merzouki A, et al T cell receptor mediated anergy of a human immunodeficiency v irus (HIV) gp120 specific CD4(+) cytotoxic T cell clone, induced by a natural HIV type 1 variant peptide. J Virol 2000; 74: 2121 30. [ 190 ] Garrison KE, Jones RB, Meiklejohn DA, et al T cell responses to human endogenous retroviruses in HIV 1 infection. P LoS Pathog 2007; 3:e165. [ 191 ] SenGupta D, Tandon R, Vieira RG, et al Strong human endogenous retrovirus specific T cell responses are associated with control of HIV 1 in chronic infection. J of virol 2011; 85: 6977 85. [ 192 ] van der Kuyl AC. HIV infect ion and HERV expression: a review. Retrovirol 2012: 9 ; 6. [ 193 ] Maksyutov AZ, Bachinskii AG, Bazhan SI, Ryzhikov EA, Maksyutov ZA. Exclusion of HIV epitopes shared with human proteins is prerequisite for designing safer AIDS vaccines. J Clin Virol 2004; 3 1: S26 38. [ 194 ] Bannert N, Kurth R. Retroelements and the human genome: New perspectives on an old relation. Proc Natl Acad Sci U S A 2004; 101: 14572 79. [ 195 ] Rolland M, Nickle DC, Deng W, et al. Recognition of HIV 1 peptides by host CTL is related to HIV 1 similarity to human proteins. PLoS One 2007; 2: e823. [ 196 ] Sacha JB, In Jeong K, Chen L, et al. Vaccination with Cancer and HIV Infection Associated Endogenous Retrotransposable Elements Is Safe and Immunogenic. J Immunol 2012; 189: 1467 79. [197 ] Neumann Haefelin C, Timm J, Schmidt J, et al Protective effect of human leukocyte antigen B27 in hepatitis C virus infection requires the presence of a genotype specific immunodominant CD8+ T cell epitope. Hepatology 2010; 51: 54 62.
103 [19 8 ] Kataria RK, Brent LH. Spondyloarthropathies. Am Fam Physician 2004; 69: 2853 60 [19 9 ] Nguyen B Y, Reveille JD. Rheumatic manifestations associated with HIV in the highly active antiretroviral therapy era. Curr Opin Rheumatol 2009; 21: 404 10. [200 ] Barber A M Hizi A Maizel JV Hughes SH. HIV 1 reverse transcriptase: structure predictions for the polymerase domain. AIDS Res Hum Retroviruses 1990; 6: 1061 72. [ 201 ] Kim B, Ayran JC, Sagar SG, et al New human immunodeficiency virus, type 1 reverse transcriptase (HIV 1 RT) mutants with increased fidelity of DNA synthesis. J Biol Chem 1999; 274 : 27666 73. [20 2 ] Kim B, Hathaway T R, Loeb L A. Human immunodeficiency virus reverse transcriptase. Functional mutants obtained by random mutagenesis coupled with genetic selectio n in Escherichia coli. J Biol Chem 1996; 271 : 4872 8. [20 3 ] Shanmugam S Madhavan V Kantor R et al Unusual Insertion and Deletion at Codon 67 and 69 of HIV Type 1 Subtype C Reverse Transcriptase Among First Line Highly Active Antiretroviral Treatment F ailing South Indian Patients: Association with Other Resistance Mutations. AIDS Res Hum Retroviruses 2012; 28: 1763 65
104 BIOGRAPHICAL SKETCH Missa Patrick Sanou was born in Bobo Dioulasso, economical capital of his native country, Burkina Faso. He had a chi ldhood filled with love, laughter, and play. The love for learning has always been with him in the natural sciences, history, politics or literature. Sports have also always been a big part of his life, the most influential being handball, basketball and soccer. He loves music and as a teenager, was member of a rap band in his hometown of Ouagadougou. In the United States, he completed his undergraduate studies, on a basketball scholarship at Armstrong Atlantic State University i n Savannah Geor gia He is now part of the Gator N ation for life! He has always been driven by the will to improve the life conditions of the most disadvantaged. As he completes his PhD studies, he looks ahead with hope to a changing world in which he believes he can have a positive influence, not only as a scientist, but just as a member of his community, to help create a better place for all.