International AIDS Society–USA Topics in HIV Medicine

HIV Pathogenesis and Development

Amitinder Kaur, MD, and R. Paul Johnson, MD

Continuing the trend of recent years, long-term nonprogression (LTNP) with HIV-specific CD8+ T cells was 5705 the 10th Conference on Retroviruses strong HIV-specific CD4+ and CD8+ T- spot-forming cells/106 peripheral blood and Opportunistic offered a responses is well-established, the mononuclear cells (PBMCs) (range, 185- strong and diverse array of presenta- factors associated with progressive HIV 25,000). Although the plasma HIV-1 tions on AIDS pathogenesis and vaccine are not well-understood. RNA levels and CD4+ T-cell counts in development. Recent advances in Using a panel of 410 overlapping this group showed a weak inverse cor- pathogenesis highlighted peptides spanning all HIV proteins of a relation with the breadth and magni- efforts to understand how HIV persists clade B consensus sequence, Kaufmann tude of the CD8+ T-cell response, a in the face of a vigorous immune and Draenert presented results of a striking overlap in the magnitude and response and underscored the impor- comprehensive investigation of the total breadth of CD8+ T-cell responses was tance of assessing functional properties HIV-specific CD4+ and CD8+ T-lym- observed between progressors and of cellular immune responses. This phocyte response in different cohorts LTNPs. Thus, in 25 LTNPs, recognition of year's conference also witnessed a of HIV-infected subjects. Kaufmann a median of 16 peptides (range, 5-58) resurgence of interest in neutralizing (Abstract 31) presented an analysis of with magnitudes ranging between 1046 antibody responses, and numerous HIV-specific CD4+ T-cell responses in and 43,000 spot-forming cells/106 efforts to translate new insights into the 32 HIV-infected subjects who either PBMCs (median, 7980) did not differ structure of the HIV-1 envelope and were on highly active antiretroviral ther- significantly from progressors. neutralizing antibodies to the develop- apy (HAART) or were off therapy and Since T- responses to HIV ment of new vaccine approaches able to had relatively low plasma HIV-1 RNA have been routinely measured using induce broadly neutralizing antibodies. levels (median, 2850 copies/mL). HIV- nonautologous viral sequences, the real Nonhuman primate vaccine studies specific CD4+ responses were focused breadth and magnitude of the HIV-spe- offered encouraging results of novel on relatively few epitopes, predomi- cific T-cell response as gauged by autol- approaches able to induce protection nantly found in Gag and Nef. CD4+ T- ogous sequences is likely to be against pathogenic simian immunodefi- cell responses to at least 1 HIV peptide even greater. Using peptide sets from a ciency virus (SIV) isolates but also were detected in 78% of patients, and a clade B consensus sequence, Altfeld and offered concerning caveats regarding median of 4.5 peptides were recognized colleagues (Abstract 314) showed that the potential for viral escape and the by each subject. Despite the presence of the virus-specific T-cell response clus- impact of host genotype on vaccine pro- disparate human leukocyte tered in conserved regions of the tection. (HLA) class II alleles, 7 peptides (5 in . However, when responses to Gag and 2 in Nef) were recognized by peptides spanning the consensus and Immune Control of HIV: Size is 75% of the subjects, including 1 that autologous virus sequences of Tat, Vpr, Not Enough was recognized in 56% of subjects. This and p24-Gag were compared, the autol- finding suggests a relatively promiscu- ogous sequences demonstrated broader In the ongoing quest for an effective ous presentation of CD4+ T-cell epi- and significantly stronger responses, AIDS vaccine, advancing our under- topes by different HLA class II alleles. In particularly those directed against the standing of the barriers that impede contrast to the CD4+ T-cell response, variable regulatory proteins. Thus, dif- immune control of viral replication in the CD8+ T-cell response in this cohort ferences between controllers and non- HIV-infected individuals is of paramount of HIV-infected subjects was 10-fold controllers of HIV infection may be bet- importance. Although the association of greater in magnitude and broadly ter appreciated using autologous virus directed, targeting reverse transcriptase sequences. However, Migueles (Abstract Dr Kaur is Instructor in Medicine at (RT), integrase, and Env in addition to 318) showed that the frequency of the Division of , New Gag and Nef. A similar pattern of vigor- CD8+ T secreting interfer- England Regional Primate Center, ous and broad HIV-specific CD8+ T-cell on-γ in response to stimulation with Harvard Medical School, in South- responses was also observed by autologous virus-infected CD4+ T cells borough, Mass. Dr Johnson is Associate Draenert (Abstract 35) in 26 chronically was equivalent in LTNPs and progres- Professor of Medicine at the Division HIV-infected subjects with late-stage sors. In addition, variant peptides with of Immunology, New England HIV infection (mean plasma HIV-1 RNA autologous virus sequences were well- Regional Primate Center, and at the level, 186,271 copies/mL, and mean recognized, demonstrating that loss of Infectious Unit and Partners CD4+ count, 141 cells/µl). A median of immune control can occur in the pres- AIDS Research Center, Massachusetts 13.5 peptides (range, 2-39) were recog- ence of persistent recognition of autolo- General Hospital, Harvard Medical nized by individual progressors in this gous virus sequences. School, in Charlestown, Mass. group, and the median magnitude of These data highlight the paradox

76 Conference Highlights - HIV Pathogenesis and Vaccine Development Volume 11 Issue 3 May/June 2003

that despite the fact that HIV infection is represent novel CD8+ T-cell epitopes in marked inability to proliferate in highly immunogenic, induction of a the autologous virus or sites of escape response to antigen-specific stimulation strong and broad anti-HIV response is from CD4+ T lymphocytes or anti- in progressors but not in LTNPs. This not in itself sufficient for effective bodies. defect in proliferative capacity was immune control. This finding raises the Loss of immune control in the set- linked to a lack of perforin expression in possibility that factors other than the ting of a strong and broad HIV-specific the nonproliferating antigen-specific overall magnitude of cellular immune T-lymphocyte response was also CD8+ T cells. A similar defect in prolif- responses may be critical in the ability observed in subjects undergoing struc- erative capacity has been seen in HIV- of the host to control HIV infection. Are tured treatment interruptions (STIs) fol- specific CD4+ T cells in individuals on there distinct functional properties of lowing institution of HAART in acute STI during viremic episodes. the HIV-specific T-cell response associat- HIV infection. Walker presented an Price and colleagues (Abstract 32) ed with an effective ? update on 14 such subjects. Substantial compared the physical enumeration of Presentations by Walker (Abstract 164) augmentation of cellular immunity was HIV-specific CD8+ T lymphocytes by and Connors (Abstract 165) addressed observed following each episode of STI. tetramers or by clonotypic quantitative this key issue. In 1 individual, the number of peptides polymerase chain reaction for the T-cell The ability of cytotoxic T lympho- recognized increased from 2 pre-STI to receptor B (TCRB) locus of defined HIV- cytes (CTLs) to control HIV replication 25 at the end of the second STI. Post-STI specific CTL clones with functional enu- may be dependent on a variety of fac- responses were also broader, encom- meration of interferon-γ producing cells tors, including genetic background, passing both structural and regulatory during viremic and aviremic episodes in presence of CTL escape, and defects in proteins. Although the pre-STI respons- 133 HIV-infected subjects enrolled in CTL maturation or function. In his pre- es were exclusively in Gag, Env, or Nef, the Swiss HIV Cohort Study. In this sentation, Walker (Abstract 164) pro- following STI, 100% of the responses study, individuals were subjected to posed that one of the reasons underly- targeted Nef, Env, and Pol; 88% target- cycles of 8 weeks on and 2 weeks off ing lack of effective viral control with ed Gag; and 38% to 60% were directed therapy, and treatment was discontin- strong CTL responses may be related to toward regulatory proteins. However, ued after 4 such cycles. During “off- epitope-specific or allele-specific quali- even this augmentation in breadth was treatment” phases, increases in plasma tative differences among CTLs. These not sufficient to sustain inhibition of HIV-1 RNA levels were associated with variations may lead to differences in the viral replication, and eventually decline in interferon-γ ELISPOT respons- propensity for and the consequences of immune control was lost. No benefit in es in the presence of stable or increased CTL escape. Thus, loss of certain CTL survival was observed in an intent-to- frequencies of tetramer-positive cells. In responses may be more important than treat analysis. However, there was some contrast, the frequency of tetramer-pos- loss of others. One such example is the benefit in terms of plasma HIV-1 RNA itive and interferon-γ-positive cells consequence of CTL escape in a single levels, in that the time to reach greater approximated each other in aviremic B27-restricted epitope (KK10). Although than 30,000 HIV-1 RNA copies/mL in individuals. Functional and nonfunc- the presence of HLA-B27 is highly asso- plasma was slowed in individuals on tional CD8+ T cells had the same clon- ciated with nonprogression in HIV- STIs. ality and were not preferentially infect- infected individuals, escape in this par- What qualitative defects are corre- ed with HIV. ticular CTL epitope is strongly correlated lates of poor immune control? In studies A novel method of assessing the with loss of immune control of viral presented by Connors (Abstract 165), function of CD8+ T cells was presented replication. Often, factors other than reduced proliferative capacity of HIV- by Betts and colleagues (Abstract 306). sequence variation in known CTL epi- specific CD8+ T cells was the single They assessed degranulation of cyto- topes appear to be responsible for non- most dominant defect that differentiat- toxic granules in CTLs by flow-based control. Thus, in a noncontroller, only 4 ed progressors from nonprogressors, as measurement of lysosomal-associated of 13 peptide responses identified by well as distinguishing viremic and membrane glycoproteins CD107a, enzyme-linked immunospot (ELISPOT) aviremic episodes in HIV-infected sub- CD107b, and CD63. These proteins are assays differed from the viral sequence jects on intermittent HAART. The HLA- expressed only on the membranes of of the autologous virus. In longitudinal B*5701 allele was present in 15 of 17 cells that have recently undergone studies of sequence variation in the patients in a cohort of LTNPs. The mag- degranulation following antigen-specific whole HIV genome in 2 HIV-infected nitude of the HIV-specific CD8+ T-cell stimulation, and may serve as a surro- subjects, Walker showed that although response, as assessed by interferon-γ gate marker for cytolytic ability of anti- increasing sequence variation with time secretion, did not differ between HLA- gen-specific CD8+ T lymphocytes. was associated with increasing plasma B*5701-positive and -negative progres- Functional heterogeneity with regard HIV-1 RNA levels, amino acid variation sors. Neither did it differ between HLA- to cytokine-secreting and degranulating in as few as 15 amino acids of all HIV B*5701-positive progressors and non- ability existed within CD8+ T-cell proteins was temporally associated with progressors. The difference in immune clonotypes specific for a single antigen. the loss of immune control. In both indi- control between the 2 groups was not How lack of function demonstrated by 1 viduals, more than half of the amino due to escape of HLA-B57-restricted CTL technique correlates with lack of acid changes were not at sites of previ- epitopes from immune control. Instead, function by other techniques (eg, ously defined CTL epitopes. These may HLA-B57-restricted CTLs showed a decreased cytokine secretion vs defec-

77 International AIDS Society–USA Topics in HIV Medicine

tive granulation vs decreased perforin (Abstract 30) analyzed the frequency induces widespread immune activation vs decreased proliferative ability) and phenotype of SIV-specific CD8+ T and dysfunction that affects numerous remains to be determined. lymphocytes targeting the Mamu-A*01- arms of the . In a ple- Phenotypic differences among anti- restricted SIV Gag181-189 epitope in the nary lecture, Fauci (Abstract 119) out- gen-specific T lymphocytes that reflect peripheral blood and reproductive tis- lined results from studies on the effect distinct stages of maturation have been sues of female SIV-infected rhesus of HIV viremia on CD4+ T lympho- correlated with their functional ability. macaques. The frequency of tetramer- cytes, B lymphocytes, and natural Harari and colleagues (Abstract 33) positive cells was increased an average killer (NK) cells performed on HIV- detected CD45RA+ CCR7– HIV-specific of 14-fold in genital mucosa compared infected individuals during viremic and CD4+ T lymphocytes in LTNPs but not with that in peripheral blood. Mucosal aviremic stages of infection. In viremic in progressors and observed an inverse CD8+ T lymphocytes had a high fre- subjects, a number of abnormalities in correlation between viremia and fre- quency of CXCR3+ cells compared CD4+ cells, B cells, and NK cells were quency of CD45RA+ CCR7– HIV-spe- with peripheral blood. The increased detected. Resting CD4+ T lympho- cific CD4+ T lymphocytes. These dif- expression of CXCR3 on vaginal CD8+ cytes in viremic subjects had sponta- ferences appeared to be restricted to T lymphocytes correlated with expres- neous virus production, whereas those HIV-specific T lymphocytes, since sion of its ligand CXCL9 in lymphoid in aviremic subjects did not. Microarray cytomegalovirus (CMV)-specific CD4+ aggregates and lamina propria of the analysis revealed upregulation of 493 T lymphocytes with a CD45RA+ vaginal mucosa, and may be a mecha- genes involved in transcriptional regula- CCR7– phenotype were present in both nism for homing of antigen-specific T tion and RNA processing in resting LTNPs and progressors. Complementary lymphocytes to mucosal sites of viral CD4+ T cells from viremic subjects. findings for CD8+ T cells were replication. Fauci proposed that the effect of observed by Draenert (Abstract 35), The inability of most HIV-infected viremia on CD4+ T cells appears to be who showed an association between subjects to generate potent neutralizing mediated by both cytokines and the HIV the presence of increased frequencies antibodies effective against primary HIV envelope. of CD45RA+ CCR7– (mature effector isolates has led many to question Effects of viremia were also demon- phenotype) HIV-specific CD8+ T cells whether antibodies play a significant strated on B cells and NK cells. B lym- and viral control. role in controlling viral replication. phocytes in HIV-viremic subjects were Boutboul and colleagues (Abstract Shaw (Abstract 166) described analysis characterized by loss of CD21 expres- 34) used complementary DNA (cDNA) of neutralizing antibody responses sion and decreased proliferation in microarray analysis to compare gene using an assay in which autologous response to CD4+ T-cell help due to expression in PBMCs from 5 healthy envelope sequences are used to gener- downregulation of CD25 expression. donors and 27 HIV-infected subjects at ate pseudotyped HIV particles express- As reported in more detail in a subse- different stages of disease. Interferon-α ing a reporter gene. This process in turn quent oral presentation by Malaspina and perforin genes were upregulated, allows analysis of neutralizing antibody (Abstract 128), B cells from HIV-viremic while the interleukin-7 receptor (IL-7R) titers to autologous viral sequences subjects have a reduced capacity to gene was downregulated in resting using a single-round infectivity assay. stimulate CD4+ T cells through PBMCs from HIV-infected individuals Using this assay, he was able to demon- CD80/86-CD28 interactions. B cells of with late-stage disease compared with strate emergence of autologous neutral- HIV-infected viremic subjects had PBMCs from HIV-seronegative individu- izing antibodies as early as 72 days decreased induction of CD80/86 on acti- als. The degree of upregulation or down- after seroconversion and titers that vated B cells and a reduced capacity to regulation of these genes was compara- reached as high as 2500. However, vari- induce proliferation of allogenic CD4+ ble to that observed in normal PBMCs ant viral sequences resistant to concur- T cells from healthy HIV-seronegative activated in vitro by CD3/CD28 stimula- rent neutralizing antibodies rapidly subjects. This functional abnormality tion. Flow cytometric analysis revealed emerged, such that neutralizing anti- was corrected following reduction of that CD8+ T lymphocytes with high body titers to autologous envelope plasma HIV-1 RNA levels by effective CD127 (IL-7R) expression were negative sequences at the time virus was isolated antiretroviral therapy and was associat- for perforin, and reciprocally, that cells were relatively low. Analysis of ed with upregulation of CD80/86 on the expressing high levels of perforin were sequence changes associated with the B lymphocytes. NK cells from viremic CD127-negative or -low. These findings of escape supported HIV-seropositive subjects had a reduced suggest the possibility that low expres- the concept of a "glycan shield," in ability to secrete beta- and sion of IL-7R on CD8+ T lymphocytes which an ever-shifting umbrella of sugar suppress HIV replication in vitro. The in HIV infection may represent cells that residues blocks the ability of antibodies activating NK receptors NKp46, NKp30, are lytic but that have lost their ability to to bind to envelope oligomers. NKp44, and NKG2D were markedly proliferate. reduced, and some killer inhibitory An additional determinant of Effect of Viremia on Immune receptors were upregulated on NK cells immunologic control is the presence of Function of viremic as opposed to aviremic HIV- adequate numbers of antigen-specific T infected subjects. lymphocytes at tissue sites of viral repli- It is now abundantly clear that in addi- A novel mechanism of viremia- cation. Cromwell and colleagues tion to depletion of CD4+ T cells, HIV induced immune evasion was proposed

78 Conference Highlights - HIV Pathogenesis and Vaccine Development Volume 11 Issue 3 May/June 2003

by Brainard and colleagues (Abstract infection, was linked to decreased sur- morphisms was strongly predictive of 29). Previous work from this laboratory vival and increased risk of AIDS-associ- plasma HIV-1 RNA levels, with greater had demonstrated that the ated dementia once HIV infection was viral adaptation being associated with SDF-1, while serving as a chemoattrac- established. The deleterious effect of higher HIV-1 RNA level. Although uni- tant at low concentrations, was able to this polymorphism was possibly related variate analysis showed a significant mediate repulsion of T lymphocytes to increased of MCP-1 and association between the presence of (termed fugetaxis) in a CXCR4-receptor- increased recruitment to the HLA B*5701 or HLA-B*2705 and low mediated manner at higher concentra- brain. Kaslow (Abstract 56) summarized plasma HIV-1 RNA levels, this effect was tions. Their current data suggest that the current knowledge of host genetic no longer apparent on multivariate increased concentrations of gp120 may polymorphisms that have consistently analysis. Instead, low plasma HIV-1 induce fugetaxis of HIV-specific CTLs. been shown to have an effect on HIV RNA level was strongly associated with Fugetaxis induced by gp120 was CD4- transmission or disease progression. At less HIV adaptation. It is possible that independent and inhibited by anti- the level of HLA alleles, the most con- the protective effect of HLA B*5701 and CXCR4 and pertussis toxin. By altering sistent deleterious effects on disease B*2705 is causally related to a cell density and performing CTL assays progression have been seen with HLA decreased propensity for their CTL epi- in flat-bottomed wells, these investiga- homozygosity at any locus and with the topes to accrue mutations because of tors demonstrated decreased efficacy of presence of certain allelic variants of the presence of genetic barriers. cell killing at lower cell densities, sug- HLA-B35 and HLA-B*5301. In contrast, Beneficial and deleterious effects of gesting the possibility that CTL migra- alleles B*5701 and B*5703 are consis- HLA class I haplotypes on HIV may also tion may affect killing efficacy. tently associated with control and be mediated via its effect on innate According to this scenario, gp120-medi- delayed progression of HIV-1 infection immunity. Carrington (Abstract 55) pre- ated inhibition of CTL migration toward across HIV subtypes A, B, and C. With sented data on associations between sites of increased viral replication may regard to impact on HIV acquisition, killer immunoglobulin-like receptors reduce CTL efficacy in vivo. sharing of HLA alleles by transmitter (KIRs) and HLA that can modulate HIV- and recipient at 1 or more loci can be 1 disease. The KIR genes, located on Host Genetics associated with an increased risk of hor- chromosome 19q13.4 in the leukocyte izontal transmission. In a study of 221 receptor complex, encode a group of Host genetic polymorphisms can affect serodiscordant couples in Zambia fol- receptors that regulate inhibition and the outcome of HIV infection at the level lowed up for 5 years, there was a strong activation of NK cells. Binding of of virus entry, virus dissemination, and deleterious effect on transmission of inhibitory KIR receptors on NK cells to immune control of viral replication. In a sharing alleles at the HLA-B locus. The major histocompatibility complex symposium, 4 presentations discussed effect of the HLA-A locus on clinical out- (MHC) class I molecules on target cells the impact of host genetics on HIV-1 come or HIV transmission has not been inhibits NK-mediated cytolysis, whereas variability and outcome of HIV infec- consistent across studies. The HLA- the absence of MHC class I molecules tion. In large population studies, Ahuja A2/A*6802 supertype has been associ- on target cells is associated with NK cell (Abstract 53) presented data on the ated with a decreased risk of horizontal activation and killing. The presence of 1 impact of genetic variation in the CCR5 and mother-to-child transmission in activating KIR allele, KIR3DS1, in com- gene and MCP-1 gene on HIV-1 trans- white and African American popula- bination with a specific subset of HLA-B mission and disease progression. In a tions infected with clade A or B virus. alleles (those containing the Bw4 sero- cohort of 649 children exposed perina- Surprisingly, the same alleles were logical epitope that also have an tally to HIV and followed for 4 years, shown to be significantly associated isoleucine at position 80, designated as multiple polymorphisms in the CCR5 with an increased risk of transmission HLA-B Bw4-80I), was shown to have a gene other than the well-described ∆32 in a Zambian cohort infected with clade highly protective effect against progres- genotype were identified that were C virus. sion to AIDS. This protective effect was associated with either increased or The effect of HLA polymorphisms in not observed with HLA-B Bw4-80I in the decreased risk of mother-to-child trans- driving HIV-1 sequence variability was absence of KIR3DS1. Further, KIR3DS1 mission. CCR5 polymorphisms that elegantly presented by John (Abstract alone shows a deleterious effect on sur- were either deleterious or beneficial had 54). HIV adapts to the host at 2 levels. At vival. HLA molecules expressing the a similar impact on disease progression 1 level the accumulation of mutations is Bw4 serologic epitope are a ligand for in both adults and children. The preva- driven by immune pressure, which for the inhibitory KIR receptor encoded by lence of disease-modifying CCR5 haplo- CTL epitopes is evident as HLA class I KIR3DL1. Since the extracellular types was different in populations of dif- allele-specific polymorphisms at the domains of 3DL1 and 3DS1 are very ferent ethnicities, emphasizing race as a population level. However, the emer- similar, they may interact with similar confounding factor in therapeutic effica- gence of mutations at each residue is ligands. The model to explain the pro- cy studies. Two polymorphisms and 3 modulated by variable genetic barriers, tective synergistic effect of HLA-B Bw4- haplotypes were also identified in the including functional or structural con- 80I and KIR3DS1 proposes that MCP-1 gene. A 2578G single-nucleotide straints or cost to replicative fitness. In a KIR3DS1 may preferentially bind to polymorphism, although associated full-length HIV genome analysis in 175 Bw4-80I, leading to NK cell activation with a decreased risk of acquiring HIV patients, adaptation of HIV to HLA poly- and killing of HIV-infected target cells.

79 International AIDS Society–USA Topics in HIV Medicine

Swift NK cell activation mediated with Vpr, investigators showed that in marked declines in NKT cells, which through KIR molecules early in infection monocyte-derived DCs, HIV was recruit- may be mediated by direct infection. may positively impact viral control, not ed to sites of cell contact with T cells. At Decline in CD4+ NKT cells but not total only for HIV, but also for other the same time, in the target cell, CD4 NKT cells correlated inversely with plas- . and CXCR4 were concentrated at the ma HIV-1 RNA level. CD4+ /DC interface. The localized Dendritic Cells concentration of HIV, CD4, and core- Natural Hosts of SIV Infection ceptors (termed an “infectious Although DC-SIGN, a C-type lectin that synapse”) may play a critical role in An expanding number of nonhuman is highly expressed in monocyte- facilitating infection of T cells by den- primates such as sooty mangabeys and derived dendritic cells (DCs), is believed dritic cells. African green monkeys serve as nat- to be a key molecule facilitating dendrit- ural hosts for SIV infection. Despite life- ic cell-mediated HIV infection of CD4+ Innate Immunity long infection and levels of viremia that T cells, the precise mechanisms under- can in some equal or exceed lying this function are not known. In addition to acting as a conduit for those in HIV-infected subjects with KewalRamani (Abstract 110) summa- HIV transmission, DCs are the principal AIDS, these hosts remain asymptomatic rized research on DC-SIGN from his lab- antigen-presenting cells in the body and do not develop AIDS. Increasing oratory. Stable expression of DC-SIGN in and essential for mounting an effective attention over the past several years the monocytic cell line THP-1 resulted adaptive immune response. Loré and has been devoted to trying to eluci- in transmission of HIV-1 at efficiencies colleagues (Abstract 81) demonstrated date immunologic or virologic mecha- similar to those of primary immature that innate immune recognition medi- nisms associated with the lack of DCs. In transwell experiments, cell con- ated by binding of the microbial pat- pathogenicity of primate lentiviruses in tact between THP-1/DC-SIGN cells and tern recognition Toll-like receptors their natural hosts. Barry and colleagues CD4+ T lymphocytes was required for (TLRs) on DCs to their natural ligands (Abstract 120) proposed that a limited HIV-1 transmission. When other trans- resulted in maturation of DCs and SIV-specific CD8+ T-cell response may formed cell lines (K562 and 3T3) stably enhancement of adaptive virus-specific play a causal role in the lack of immun- transfected and expressing DC-SIGN at immune responses in vitro. CD11c+ odeficiency in sooty mangabeys natu- levels comparable to those of the THP- myeloid DCs expressing TLRs 3, 4, and rally infected with SIVsm. Analysis of 1/DC-SIGN cells were examined, they 7 and CD123+ plasmacytoid DCs several markers of T-cell activation (eg, were not able to transmit HIV-1, even expressing TLRs 7 and 9 were activated CD69, CD25, and HLA-DR) or cell prolif- though their ability to bind HIV-1 or on exposure to their respective ligands: eration (Ki67) revealed only minor intracellular adhesion molecule 3 CpG (TLR9 receptor), imidazo- increases in SIVsm-infected sooty (ICAM3) was not impaired. This finding quinolones (TLR7), LPS (TLR4), and mangabeys compared with those led to a search for cell molecules poly I:C (TLR3). TLR-ligand-mediated observed in uninfected mangabeys. required for DC-SIGN-mediated HIV activation of plasmacytoid or myeloid Similar findings were observed even in transmission. As observed for immature DCs resulted in an enhanced capacity the setting of acute SIVsm infection of DCs, HLA-DR and leukocyte function to generate antigen-specific T lympho- sooty mangabeys, whereas acute infec- antigen 1 (LFA-1) were expressed on cytes. The ability to modulate adaptive tion of rhesus macaques with the same THP-1 cells permissible for HIV trans- immunity makes TLR ligands a poten- stock of SIVsm resulted in significant mission, but these molecules were not tially useful immunologic adjuvant for increases in T-cell activation, increased expressed on K562 and 3T3 cells. HIV . proliferation of CD8+ and CD4+ T However, expression of HLA class II Unutmaz (Abstract 112) outlined the cells, and progression to AIDS. molecules and LFA-1, singly or in com- role of natural killer T cells (NKT cells) in Depletion of CD8+ T cells using a bination, in the nonpermissive K562 HIV infection. NKT cells are a distinct murine CD8-specific monoclonal anti- and 3T3 cell lines did not reverse the subset of human effector T lymphocytes body resulted in only 3-fold increases in defect in HIV transmission, suggesting that express an invariant T-cell receptor plasma viremia, leading Barry and col- that DC-SIGN expression is not suffi- and recognize glycolipid pre- leagues to conclude that a weak or cient for efficient HIV-1 transmission sented by the nonpolymorphic MHC absent SIV-specific CD8+ T-cell and that other cofactors are likely to class I molecule CD1d. Using flow response may be an important factor in play a role. Identification of these spe- cytometry to identify NKT cells in the lack of immunodeficiency in SIV- cific cofactors should provide valuable humans (Vα24+Vβ11+ or CD1d infected sooty mangabeys. insights into DC-SIGN-mediated trans- tetramer+Vβ11+), 20% to 90% of A contrasting view was reported by mission of HIV. NKT cells were found to be CD4+ and Kaur (Abstract 121), who described the Studies using live-cell video to express CCR5. Autologous DCs results of a study in which CD8+ T cells microscopy to examine transfer of HIV pulsed with the glycolipid alpha-galacto- were depleted in SIV-infected sooty from DCs to CD4+ T lymphocytes were sylceramide were able to expand NKT mangabeys using a chimeric mouse- presented by Hope (Abstract 113). cells, which secreted IL-4 and IL-5 and human CD8-specific monoclonal anti- Using fluorescent labeling of HIV by were highly susceptible to infection with body (cM-T807). This antibody has been incorporation of a fusion protein of GFP R5-tropic HIV. HIV-infected subjects had widely used in nonhuman primate

80 Conference Highlights - HIV Pathogenesis and Vaccine Development Volume 11 Issue 3 May/June 2003

experiments and generally induces Describing one approach to better these intermediate forms of envelope, more prolonged and complete deple- understand why it has been so difficult thereby blocking virus entry, has been tion of CD8+ T cells than has been to induce broadly neutralizing antibod- controversial. Weiss (Abstract 185) obtained with murine monoclonal anti- ies by , Burton discussed reviewed efforts to generate antibodies bodies. Following a depletion of CD8+ detailed structural studies of 2 neutraliz- able to block viral fusion. She identified T cells that often extended to 3 weeks, ing monoclonal antibodies, b12 and 3 distinct stages at which fusion Kaur and colleagues observed up to 2G12. The first is a well-characterized inhibitors may inhibit virus entry: pre- 100-fold increases in plasma viremia in neutralizing antibody that was initially vention of conformational changes, 5 of 6 animals studied, whereas no sig- isolated by a phage display library blocking close opposition of the virus nificant increases in viremia were derived from an asymptomatic HIV- and host , and preven- observed in 4 animals that received a infected individual. Previous work had tion of fusion pore assembly. One of the control chimeric monoclonal antibody. demonstrated that b12 recognized a intermediate steps of viral entry Further evidence for SIV-specific CD8+ conserved site of gp120 that overlaps involves conformational changes in the T-cell responses was provided by the with the CD4 binding site. A subsequent ectodomain of gp41, in which 2 heptad finding of significant ELISPOT respons- analysis of the crystal structure of b12 repeats of gp41 are brought together to es to SIV proteins in most SIV-infected revealed a long protrusion at the tip of make up the prehairpin intermediate. mangabeys. Reasons for the apparent the antibody that inserts into the This hairpin intermediate subsequently discrepancy in findings between these recessed CD4 binding site of gp120. In generates a 6-helix hairpin structure groups was not immediately clear, but an effort to enhance production of anti- that catalyzes formation of the fusion may relate to differences in the efficacy bodies with similar structure to b12, pore. Peptides mimicking the gp41 hep- of CD8+ T-cell depletion induced by the Burton and colleagues have tried to tad repeats are able to potently inhibit 2 different monoclonal antibodies. block production of antibodies to other HIV replication, which suggests that common epitopes on the gp120 antibodies might also be able to block Prophylactic AIDS Vaccines oligomer by hyperglycosylation of this critical step in virus entry. Inherent regions such as the CD4 binding site challenges to this approach include ster- Neutralizing Antibody Responses and the V3 loop. studies ic hindrance (ie, whether antibodies can with the hyperglycosylated gp120 fit in the small gap between the virus After several years of being overshad- molecule are in process. A similar struc- particle and the host cell membrane) owed by research on cell-mediated tural analysis has been carried out for and whether sufficient time exists dur- immune responses to HIV, research on the 2G12 antibody, which recognizes an ing viral fusion to allow the antibody to neutralizing antibodies has recently epitope in the C4/V4 region of gp120 bind. Immunization with recombinant enjoyed a renaissance prompted by and has been previously shown to rec- immunogens that mimic either the pre- improved information on the structural ognize a cluster of oligomannose gly- hairpin or 6-helix bundle conformation basis for neutralization, new assays for cans in this region of gp120. Crystal of gp41 resulted in antibodies able to measuring neutralizing antibodies, and structure analysis of 2G12 revealed that bind either early intermediates or late continued hope that structurally modi- instead of forming the typical Y shape intermediates, respectively, and that fied envelope immunogens will of most (IgG) were able to inhibit viral entry. These enhance the induction of broadly reac- molecules, the hypervariable heavy data provide an important proof-of-prin- tive neutralizing antibodies to HIV. chains (VH) formed a tight cluster in ciple demonstration of the feasibility of

Leading off a symposium devoted sole- which the 2 VH domains are directly jux- inducing antibodies able to bind to ly to neutralizing antibodies, Burton taposed, a phenomenon called domain these fusion intermediates. (Abstract 184) reviewed the evidence swapping. The unusual structures of Generation of antibodies able to bind that supports the view that neutralizing both b12 and 2G12 provide at least 1 the chemokine coreceptor binding site antibodies represent the small fraction reason for the difficulties in generating of gp120 has been another leading of envelope-specific antibodies that are broadly neutralizing antibodies against approach for the induction of broadly able to bind the oligomeric envelope HIV. Whether this understanding will neutralizing antibodies against HIV-1. spike. According to this viewpoint, ultimately facilitate elicitation of such Sodroski (Abstract 186) described broadly reactive neutralizing antibodies antibodies by the use of modified recent efforts to define the structural are therefore the subset of antibodies immunogens will be a major challenge characteristics of antibodies able to that are able to bind to conserved sites for research in this area for the next sev- bind the chemokine receptor binding on the oligomer. Burton also noted that eral years. site. Two specific regions of gp120 although passive transfer of neutralizing After binding to cellular receptors, make up the chemokine receptor bind- antibodies in animal models has pro- gp120 undergoes a series of conforma- ing site: the variable V3 loop and the vided compelling proof-of-principle tional changes ultimately resulting in beta19 strand, a more conserved region demonstration of the ability of neutral- insertion of an oligomeric form of gp41 of the bridging sheet that is protected izing antibodies to protect against pri- into the host cell membrane and subse- by the V3 loop. Because the length of mate lentiviruses, efforts to elicit broad- quent fusion of the viral particle with IgG molecules (approximately 115 ly neutralizing antibodies by vaccina- the host cell membrane. Whether anti- angstroms) is significantly longer than tion have consistently met with failure. bodies can be successfully generated to that of the space between gp120 and

81 International AIDS Society–USA Topics in HIV Medicine

the host cell membrane after initial Nonhuman Primate Studies: in macaques immunized with a recom- virus attachment (approximately 85 The Good News binant replication-defective adenovirus angstroms), antibodies able to interact vector expressing SIV Gag either alone with the chemokine receptor binding Much of the newfound optimism in the or when given following DNA priming. site need to have a flexible and extend- AIDS vaccine field over the past 2 years In this initial study, to facilitate evalua- ed region to reach beyond the adjacent has arisen as a result of nonhuman pri- tion of cellular immune responses using V3 and V1/V2 loops. Indeed, these are mate studies employing the simian- MHC tetramers, all macaques were the characteristics of one of the best- human immunodeficiency virus (SHIV) selected to express a specific MHC class characterized chemokine receptor 89.6p as a challenge strain. Although I allele (Mamu-A*01). Although these binding antibodies, 17b. Another dis- this strain is pathogenic and results in results were clearly encouraging, ques- tinctive characteristic of many rapid CD4+ T-cell depletion over a mat- tions were raised as to whether they chemokine receptor binding antibodies ter of weeks, concerns have been could be replicated using other chal- (although not 17b) is the sulfation of raised that because of its atypical dis- lenge stocks and in animals with a more tyrosine residues in the antigen bind- ease course and ease of neutralization, diverse MHC background. In a late- ing site. As described in more detail in 89.6p may not be predictive of protec- breaker abstract (Abstract 85lb), Shiver the presentation by Farzan (Abstract tion against clinical HIV isolates. Two presented follow-up studies in 2 groups 28), this sulfation mimics the post- presentations highlighted vaccine of macaques to address these questions. translational modification of tyrosine approaches able to induce partial pro- The first study involved Mamu-A*01- residues of CCR5 that interact with pos- tection against challenge with isolates negative macaques that were immu- itively charged residues of gp120 and from pathogenic SIV of macaques nized with adenovirus vectors encoding thus play a critical role in gp120-CCR5 (SIVmac), against which it has been either SIV Gag, a heterologous envelope interactions. historically quite difficult to induce (JRFL), a homologous envelope (89.6p), Despite the disappointing failure of protection. or the combination of Gag and the het- efforts in the 1980s to generate neu- Robert-Guroff (Abstract 77) des- erologous envelope. Following intra- tralizing antibodies effective against cribed the results of a trial in which venous challenge with SHIV 89.6p, primary isolates by V3 loop immuniza- macaques were vaccinated mucosally immunized animals had at least a tion, there has been continued interest with a replication-competent aden- 100-fold decrease in set point viremia, in the potential utility of the V3 loop as ovirus. Animals were vaccinated with a and combined immunization with ade- a vaccine immunogen. Zolla-Pazner recombinant adenovirus expressing novirus vectors expressing Gag and Env (Abstract 107) described efforts to elic- Env and Rev administered either by appeared to give better protection than it V3-specific antibodies with a broader itself or in combination with recombi- that obtained with either antigen range of neutralization. Previous stud- nant adenoviruses expressing other SIV alone, reinforcing observations previ- ies using antibodies specific for linear proteins (Gag or Nef) or a recombinant ously made by the Robinson laboratory determinants of V3 had demonstrated subunit boost. Relatively strong cell- at Emory. Interestingly, the level of pro- that neutralization mediated by these mediated immune responses as deter- tection observed in this experiment antibodies was quite type-specific. mined by ELISPOT assays were appeared to be less complete than that However, recent work from the Zolla- observed against Gag and Env, with observed in previous Mamu-A*01-pos- Pazner laboratory has demonstrated weaker responses observed against itive vaccines. The second study that monoclonal antibodies specific for other SIV proteins. Neutralizing anti- involved immunization of Mamu-A*01- conformational determinants of V3 body responses were also observed in positive and -negative macaques with were able to mediate broader neutral- groups that received a recombinant sub- an adenovirus SIV-Gag vector with or ization of primary isolates. These data unit boost. Following intrarectal chal- without DNA priming. Following an presented a paradox: given the variabil- lenge with SIVmac251, a significant intrarectal challenge with the ity of the V3 loop, how were V3-specif- (20-fold) reduction of set-point viremia pathogenic SIVmac239 virus, Mamu- ic antibodies able to neutralize diverse was observed in animals that had been A*01-positive animals that received HIV-1 isolates? The answer appears to immunized with the SIV Env/Rev con- DNA priming and the adenovirus Gag lie in part in the structural constraints struct in combination with one of the boost had a significant 10- to 30-fold of the V3 loop. Nuclear magnetic reso- other SIV recombinants. A subset of ani- reduction in plasma viremia at 150 nance analysis of the structure of the mals was able to control viremia to days compared with controls. However, V3 loop bound to the V3-specific anti- undetectable levels. Protection correlat- this effect was not observed in Mamu- body 447 suggested at least 2 alterna- ed in part with ELISPOT responses on A*01-positive animals that received tive conformations of V3: 1 able to bind day of challenge, but other responses, adenovirus alone or in Mamu-A*01- to CCR5, the other able to bind to such as proliferative responses, neutral- negative animals that received the CXCR4. Selection pressure on the V3 izing antibody responses, and CD8+ DNA prime/adenovirus-boost regimen. loop to maintain these 2 alternative antiviral responses, may have played a Although the demonstration of a signifi- conformations may also provide suffi- role in mediating protection as well. cant protective effect on challenge with cient conservation to allow neutraliza- Previous work from Shiver and col- a pathogenic SIVmac strain in Mamu- tion by conformation-sensitive V3-spe- leagues had demonstrated impressive A*01-positive animals is encouraging, cific antibodies. protection against SHIV 89.6p challenge the absence of a clear significant effect

82 Conference Highlights - HIV Pathogenesis and Vaccine Development Volume 11 Issue 3 May/June 2003

in Mamu-A*01-negative animals is con- tle evidence to date for such an in vivo mate AIDS vaccine studies, progress cerning. These results further illustrate effect in macaque studies, Staprans reported in human clinical trials was the difficulties in interpreting challenge (Abstract 80) described results of a more modest. (The much-anticipated data derived from nonhuman primate study that raised a concern in this results from the phase 3 VaxGen study models and raise numerous questions. regard. In this study, macaques were were not reported until 10 days after Is SIVmac239 too rigorous a challenge? immunized either with a live attenuated the close of the conference). Hammer Which macaque model will ultimately varicella-zoster virus (VZV-OKA) (Abstract 108) provided a broad prove to be the best predictor of effica- expressing the SIVsmH4 envelope or overview of the current status of HIV cy in human clinical trials? Answers to with the parental VZV-OKA strain. In vaccine clinical trials. After detailing the these questions will be ultimately immunized animals, the authors numerous challenges facing the devel- dependent on results from phase 3 clin- observed relatively disappointing SIV- opment and testing of HIV/AIDS vac- ical trials involving approaches able to specific immune responses, which cines, he highlighted results from sever- induce potent cellular immune respons- consisted primarily of virus-binding al recently completed or ongoing vac- es and are thus at least 4 to 7 years antibodies and limited CTL responses cine trials. HVTN 203 examined the away. but no neutralizing antibodies. After of a third-generation a prolonged rest, animals were reboost- vector (vCP1452) that Nonhuman Primate Studies: ed and challenged rectally with expressed HIV-1 Gag and Env. Induction The Bad News SIVsmE660. A dramatic difference was of CTL responses to Gag or Env was dis- observed between the groups. Animals appointingly infrequent, occurring in Even more grounds for caution was that had been vaccinated with the VZV- only 13% and 7% of vaccines, respec- provided by oral presentations from OKA envelope vaccine developed signif- tively. This relative lack of immuno- Barouch and Staprans. Barouch icantly higher levels of plasma viremia genicity was significantly below the (Abstract 76) analyzed the frequency of and more rapid CD4+ T-cell depletion milestone that had been set for progres- escape from CTL surveillance in vacci- compared with controls immunized sion of this approach to phase 3 clinical nated macaques. These studies were with the unmodified VZV-OKA. trials in the United States. Results from prompted in part by a report that Increased rates of viral replication were the ongoing Merck vaccine trials have appeared last year that described correlated with increased percentages been more encouraging. At the highest escape in a dominant CTL epitope in of proliferating CD4+ T cells (as doses studied, 42% of subjects receiv- a single DNA-vaccinated animal that assessed by expression of the prolifera- ing a DNA gag vaccine had developed had been challenged with SHIV 89.6p. tion marker Ki67) 3 days after chal- ELISPOT responses to Gag after 4 In the present study, Barouch described lenge, although SIV-specific CD4+ T-cell . Initial trials of the breakthrough viremia and disease pro- responses were not directly measured. Merck replication-defective adenovirus gression in 3 of 4 Mamu-A*01-positive These results raise the concerning pos- vector expressing Gag are still being macaques that had been vaccinated sibility that induction of virus-specific analyzed, but preliminary results indi- with a DNA gag vaccine and challenged CD4+ T-cell responses in the absence cate that 78% of subjects receiving the with the pathogenic SIV stock of other effective immune responses highest dose of adenovirus had positive SIVsmE660. Emergence of CTL escape might accelerate instead of impede dis- ELISPOT responses after 2 doses. The mutants in multiple epitopes corre- ease progression. However, several sig- National Institutes of Health Vaccine lated with decreases in epitope-specific nificant caveats should be noted. There Research Center is pursuing a similar responses as determined by MHC was no direct measurement of virus- approach utilizing DNA priming with a tetramers, increased viremia, and specific CD4+ responses after chal- recombinant adenovirus vector boost CD4+ T-cell depletion. These results lenge. Also, the spontaneous suppres- and will utilize as immunogens a clade reinforce the frequent occurrence of sion of viremia in the control animals B Gag/Pol/Nef given in combination CTL escape in SIV-infected macaques challenged with the pathogenic with 3 different envelopes (clades A, B, previously described by other groups SIVsmE660 strain is unusual; most and C). Although results from phase 3 and suggest that initial gains in groups have observed significantly high- efficacy trials of these approaches decreasing plasma viremia by vaccina- er levels of viremia with this stock. remain several years away, these early tion may be lost over time due to viral Although this study raises important results demonstrating relatively robust escape. concerns, these results will have to be induction of cellular immune responses Over the past several years, several replicated with more intensive analysis offer some grounds for optimism. investigators have raised the question of of virus-specific immune responses As one approach to try to increase whether an ineffective AIDS vaccine before any hard conclusions regarding the immunogenicity of canarypox vec- might in fact worsen disease progres- potential adverse effects of vaccination tors, Goepfert and colleagues (Abstract sion, either by production of antibodies can be drawn. 82) examined whether increasing the that enhance instead of neutralize viral dose of the recombinant canarypox vec- replication or by induction of virus-spe- Human Clinical Trials tor vCP1452 up to 108 50% tissue cul- cific CD4+ T-cell responses that would ture infectious dose (TCID50) could serve to facilitate HIV replication. In contrast to the broad array of novel improve induction of cellular immune Although there has fortunately been lit- findings presented from nonhuman pri- responses. While previous lower doses

83 International AIDS Society–USA Topics in HIV Medicine

of this vector have been relatively well- Codon-optimized SIV gag and env genes gression of this approach to human tolerated, at the increased dose, the vast were fused to either the secreted clinical trials. Two presentations ana- majority of subjects (>85%) developed chemokine MCP-3 (which directs pro- lyzed the use of single-cycle SIV or HIV both local and systemic symptoms. No teins to the secretory pathway) or to the constructs (essentially nonreplicating improvement in interferon-γ ELISPOT β-catenin peptide (which directs pro- lentiviral vectors) as a potential vaccina- responses was observed, and the overall teins to the proteasomal degradation tion approach. Evans (Abstract 78) frequency of responding subjects pathway). Immunization of animals described results of immunization with remained disappointingly low (18- with the MCP/gag/env construct resulted a single-cycle SIV generated by intro- 24%). in increased humoral responses. duction of a nucleotide substitution in Macaques immunized with a combina- the gag/pol frameshift site and providing Novel Approaches tion of the unmodified MCP-3 and β- Gag-Pol expression in trans. A single catenin fusion proteins showed a broad intravenous of single-cycle SIV Although vaccinology in general (and range of immune responses, including in macaques resulted in peak levels of the study of adjuvants in particular) has CD4+ T-cell, CTL, and antibody 104 to 105 copies/mL of plasma viremia been criticized frequently for being a responses. After mucosal challenge with that rapidly decayed to undetectable largely empirical science, numerous SIVmac251, vaccinated animals had levels and low but significant SIV Gag- poster presentations highlighted novel significantly lower viral loads compared specific responses detected by ELISPOT approaches utilizing the rational use of with those of naive animals, a differ- and MHC tetramers. A second inocula- molecular adjuvants. Huang et al ence that was maintained out to 30 tion resulted in significantly lower levels (Abstract 396) analyzed the ability of weeks postchallenge. of viremia (presumably reflecting the the NKT cell ligand α-galactosylce- Although DNA vaccination has effect of preexisting SIV-specific ramide (α-GalCer) to serve as an adju- proved to be a very effective means to immune responses) and transient vant for DNA vaccination in mice. When induce cellular immune responses in boosting of SIV-specific cellular immune α-GalCer was administered in combina- rodents, results have been less impres- responses. Tung (Abstract 79) described tion with suboptimal doses of DNA vac- sive in macaques and even more disap- a complementary approach involving cines, a 5-fold increase in CD8+ T-cell pointing in humans. Whether these dif- production of replication-defective, responses and a 2-fold increase in ferences reflect true species-specific dif- vesicular stomatitis virus G (VSV-G)- CD4+ T-cell responses was observed. ferences or largely differences in the pseudotyped HIV vectors with a trunca- An alternative approach to increasing modes of administration is not clear. tion of the pol gene. Systemic and immunogenicity was presented by For instance, when expressed on a mil- mucosal resulted in virus- Zhang and colleagues (Abstract 397), ligram per kilogram basis, doses of DNA binding antibodies and virus-specific who created a fusion protein of CTLA4 administered to mice are significantly CTL responses. Challenge of animals and SIV Gag in order to target B7- higher than those used in comparable with the SHIVku-2 strain resulted in expressing, antigen-presenting cells studies in nonhuman primates or lower levels of viremia in vaccines as such as dendritic cells. Immunization humans. To address the issue of compared with levels in a concurrent with CTLA4/SIV Gag resulted in a 100- whether modifications in the method of control animal. These data support fur- fold increase in Gag-specific antibody administration could affect immuno- ther study of single-cycle lentiviruses as responses. A complementary approach genicity, Gardiner and colleagues a vaccine modality in nonhuman pri- was described by Ross and colleagues (Abstract 452) analyzed whether divi- mates, but it remains to be seen (Abstract 400), in which DNA constructs sion of a constant amount of DNA whether this approach will have suffi- were created that encoded fusions of among 1, 2, 3, or 4 limbs in mice cient advantages to outweigh the safety Env with C3d. C3d is a component of altered cellular immune responses as concerns regarding the use of an inte- the complement cascade that specifical- determined by interferon-γ ELISPOT grating virus for a preventive vaccine. ly binds to complement receptor 2 on B assays. Dividing a 20 µg DNA dose Evaluation of cell-mediated immune cells and therefore targets C3d fusion among 4 extremities significantly responses in HIV/AIDS vaccine trials has proteins to B cells. DNA immunizations increased cellular immune responses to increasingly relied upon assessment of with the Env-C3d complex resulted in a level similar to that provided by a sin- secretion of cytokines such as interfer- 103- to 105-fold increases in antibody gle 100 mg dose of DNA in one leg. on-γ using ELISPOT and intracellular titers compared with results of immu- These data suggest that very basic cytokine staining assays. In many nizations with DNA expressing Env issues regarding amount of DNA and instances, whether secretion of interfer- alone. Although it will be important to number of immunization sites need to on-γ accurately reflects the majority of see whether these encouraging results be more carefully addressed in nonhu- antigen-specific cells has not been well- in mice can be replicated in primates, man primate and human studies. addressed. Using 12-color flow cytome- the proliferation of these studies sug- Although live attenuated SIV vac- try, De Rosa (Abstract 405) analyzed the gests that rational approaches to opti- cines have proved to be one of the range of cytokines secreted by antigen- mizing immunogenicity may ultimately most effective means to induce protec- specific cells following either tetanus or bear fruit over the next several years. tion against infection with pathogenic immunization. Remarkably, A third approach to antigen targeting SIV isolates in nonhuman primates, cells secreting interferon-γ represented was reported by Rosati (Abstract 448). safety concerns have precluded pro- only a small minority of the antigen-

84 Conference Highlights - HIV Pathogenesis and Vaccine Development Volume 11 Issue 3 May/June 2003

specific response. With fresh cells, IL-2 in stimulation index and at least an or to receive HAART in combination was the dominant response, whereas increase of 500 counts per minute in at with vaccination with ALVAC HIV with frozen cells, cells producing MIP-1β least 2 antigens on numerous occa- vCP1433 (a recombinant canarypox were the dominant response, exceeding sions), a significant increase in HIV-spe- expressing HIV-1 env, gag, and nef the frequency of cells secreting interfer- cific proliferative responses, but not genes) and Lipo-6T (consisting of 6 pep- on-γ by 10-fold or more. These provoca- CD8+ T-cell responses, was observed in tides in Nef, Gag, Env, and Pol and a tive results will need to be confirmed vaccinated subjects. Unfortunately, this helper TT epitope). Five doses were with other antigens, but if validated, augmentation was not associated with administered at monthly intervals. would have significant implications for any clinical benefit. Several presenta- Following the last dose, the vaccinated evaluation of antigen-specific responses tions (Abstracts 60, 61, and 62) dis- group had a significantly higher fre- in both vaccine trials and HIV/AIDS cussed results of therapeutic vaccina- quency of subjects with detectable pro- pathogenesis studies. tion using modified Ankara liferative responses to p24 antigen. Four (MVA) or recombinant canarypox-HIV weeks after stopping HAART, 2 of 37 Therapeutic Vaccination (ALVAC) vectors in HIV-infected subjects patients in the control group and 8 of 33 on HAART. In a phase 1 trial of a MVA patients in the vaccinated group had Since immune control of HIV replication vector expressing the HIV-1 nef gene, controlled viremia. However, this effect is not achieved in most subjects, despite administration of 3 doses of the vaccine was not sustained. At present, although the presence of a readily detectable HIV- to 14 subjects on HAART with plasma some boosting of immune responses specific immune response, could thera- HIV-1 RNA levels below 50 copies/mL has been observed following therapeu- peutic augmentation of HIV-specific and CD4+ counts above 400 cells/µl tic vaccination, clear clinical benefits immunity by vaccination potentiate induced CD8+ and CD4+ T cells to have yet to be realized. existing immune responses and gener- recognize new epitopes in 10 of 16 sub- ate novel responses sufficient to induce jects (Harrer, Abstract 60). Interruption Written by Drs Kaur and Johnson in March 2003. immune control? of HAART resulted in rebound viremia Financial Disclosures: Drs Kaur and Johnson Walker (Abstract 164) presented in all subjects, although the plasma HIV- have no affiliations with commercial organi- results of a pilot using a 1 RNA levels remained lower than pre- zations that may have interests related to the whole inactivated Env-depleted vaccine HAART levels in 7 of 14 vaccinated sub- content of this article. of clade A/G Zairian isolate in an adju- jects. In a randomized control study vant to immunize chronic HIV-infected (Levy, Abstract 62), 70 patients on subjects. Using stringent criteria for aug- HAART for at least 1 year were random- Top HIV Med. 2003;11(3):76-85 mentation of immunity (5-fold increase ized to continue HAART alone (n=37) Copyright  2003 Interntaional AIDS Society–USA

85