Optimal priming of poxvirus vector (NYVAC)-based HIV vaccine regimens for T cell responses requires three DNA injections. Results of the randomized multicentre EV03/ANRS VAC20 Phase I/II Trial.

DNA vectors have been widely used as a priming of poxvirus vaccine in prime/boost regimens. Whether the number of DNA impacts qualitatively or quantitatively the immune response is not fully explored. With the aim to reinforce T-cell responses by optimizing the prime-boost regimen, the multicentric EV03/ANRS VAC20 phase I/II trial, randomized 147 HIV-negative volunteers to either 3xDNA plus 1xNYVAC (weeks 0, 4, 8 plus 24; n = 74) or to 2xDNA plus 2xNYVAC (weeks 0, 4 plus 20, 24; n = 73) groups. T-cell responses (IFN-γ ELISPOT) to at least one peptide pool were higher in the 3xDNA than the 2xDNA groups (91% and 80% of vaccinees) (P = 0.049). In the 3xDNA arm, 26 (37%) recipients developed a broader T-cell response (Env plus at least to one of the Gag, Pol, Nef pools) than in the 2xDNA (15; 22%) arms (primary endpoint; P = 0.047) with a higher magnitude against Env (at week 26) (P<0.001). In both groups, vaccine regimens induced HIV-specific polyfunctional CD4 and CD8 T cells and the production of Th1, Th2 and Th17/IL-21 cytokines. Antibody responses were also elicited in up to 81% of vaccines. A higher percentage of IgG responders was noted in the 2xDNA arm compared to the 3xDNA arm, while the 3xDNA group tended to elicit a higher magnitude of IgG3 response against specific Env antigens. We show here that the modulation of the prime strategy, without modifying the route or the dose of administration, or the combination of vectors, may influence the quality of the responses.

Recombinant fowlpox virus vector-based vaccines: expression kinetics, dissemination and safety profile following intranasal delivery.

We have previously established that mucosal uptake of recombinant fowlpox virus (rFPV) vaccines is far superior to other vector-based vaccines. Specifically, intranasal priming with rFPV vaccines can recruit unique antigen-presenting cells, which induce excellent mucosal and systemic HIV-specific CD8+ T-cell immunity. In this study, we have for the first time investigated the in vivo dissemination, safety and expression kinetics of rFPV post intranasal delivery using recombinant viruses expressing green fluorescent protein or mCherry. Both confocal microscopy of tissue sections using green fluorescent protein and in vivo Imaging System (IVIS) spectrum live animal and whole organ imaging studies using mCherry revealed that (i) the peak antigen expression occurs 12 to 24 h post vaccination and no active viral gene expression is detected 96 h post vaccination. (ii) The virus only infects the initial vaccination site (lung and nasal cavity) and does not disseminate to distal sites such as the spleen or gut. (iii) More importantly, rFPV does not cross the olfactory receptor neuron pathway. Collectively, our findings indicate that rFPV vector-based vaccines have all the hallmarks of a safe and effective mucosal delivery vector, suitable for clinical evaluation.

Safety and Immunogenicity of a Randomized Phase 1 Prime-Boost Trial With ALVAC-HIV (vCP205) and Oligomeric Glycoprotein 160 From HIV-1 Strains MN and LAI-2 Adjuvanted in Alum or Polyphosphazene.

BACKGROUND: Prime-boost regimens comprising ALVAC-HIV (prime) and human immunodeficiency virus type 1 (HIV) Env (boost) induce HIV-specific neutralizing antibody and cell-mediated immune responses, but the impact of boost schedule and adjuvant requires further definition. METHODS: A phase 1 trial was conducted. In part A (open label), 19 volunteers received oligomeric glycoprotein 160 from HIV strains MN and LAI-2 (ogp160 MN/LAI-2) with dose escalation (25, 50, 100 µg) and either polyphosphazene (pP) or alum adjuvant. In part B, 72 volunteers received either placebo (n=12) or recombinant canarypox virus expressing HIV antigens (ALVAC-HIV [vCP205]) with different doses and schedules of ogp160 MN/LAI-2 in pP or alum (n = 60). RESULTS: The vaccines were safe and well tolerated, with no vaccine-related serious adverse events. Anti-gp70 V1V2 antibody responses were detected in 17 of 19 part A volunteers (89%) and 10%-100% of part B volunteers. Use of a peripheral blood mononuclear cell-based assay revealed that US-1 primary isolate neutralization was induced in 2 of 19 recipients of ogp160 protein alone (10.5%) and 5 of 49 prime-boost volunteers (10.2%). Among ogp160 recipients, those who received pP were more likely than those who received alum to have serum that neutralized tier 2 viruses (12% vs 0%; P = .015). CONCLUSIONS: Administration of ogp160 with pP induces primary isolate tier 2 neutralizing antibody responses in a small percentage of volunteers, demonstrating proof of concept and underscoring the importance of further optimization of prime-boost strategies for HIV infection prevention. CLINICAL TRIALS REGISTRATION: NCT00004579.

Induction of rapid apoptosis for class I MHC molecule-restricted CD8(+) HIV-1 gp160-specific murine activated CTLs by free antigenic peptide in vivo.

We have previously reported that the cytotoxic activity of murine CD8(+) CTLs specific for HIV-1 gp160 envelope protein was markedly inhibited in vitro by brief exposure to a free epitope peptide P18-I10 (aa: RGPGRAFVTI) using the epitope-specific CTL line (LINE-IIIB) or a clone (RT-1). We have also shown that recently stimulated P18-I10-specific murine CTLs rapidly fell into apoptosis in vitro after brief exposure to the free epitope peptide. In the present study, we examined whether similar inactivation or apoptosis of recently stimulated CTLs occurred in vivo by exposure to the free epitope peptide using TCR transgenic (Tg-RT-1) mice expressing TCRαß genes of CTL clone RT-1. When the Tg mice were inoculated with recombinant vaccinia virus expressing HIV-1-IIIB gp160 genes followed by injection of P18-I10 epitope peptide, apparent reduction in the number of CTLs determined by flow cytometry using H-2D(d)/P18-I10 pentamer was observed within a few hours after the injection. Most of the H-2D(d)/P18-I10 pentamer-stained cells were positive for Annexin V and apoptosis was confirmed by microscopic analyses. Moreover, when mice were pretreated with immunosuppressive agents, such as cyclosporin A and tacrolimus (FK506), induction of apoptosis by P18-I10 was significantly inhibited and CTL cytotoxicity was maintained. These results suggest that the rapid loss of virus-specific CD8(+) CTLs might occur in vivo through apoptosis in the early stages of viral infection when activated CTLs may encounter viral epitope(s) released from virus-infected cells attacked by CTLs and we can prevent the loss by pretreatment with immunosuppressive agents.

Vaccine delivery to the oral cavity using coated microneedles induces systemic and mucosal immunity.

PURPOSE: The objective of this study is to evaluate the feasibility of using coated microneedles to deliver vaccines into the oral cavity to induce systemic and mucosal immune responses. METHOD: Microneedles were coated with sulforhodamine, ovalbumin and two HIV antigens. Coated microneedles were inserted into the inner lower lip and dorsal surface of the tongue of rabbits. Histology was used to confirm microneedle insertion, and systemic and mucosal immune responses were characterized by measuring antigen-specific immunoglobulin G (IgG) in serum and immunoglobulin A (IgA) in saliva, respectively. RESULTS: Histological evaluation of tissues shows that coated microneedles can penetrate the lip and tongue to deliver coatings. Using ovalbumin as a model antigen it was found that the lip and the tongue are equally immunogenic sites for vaccination. Importantly, both sites also induced a significant (p < 0.05) secretory IgA in saliva compared to pre-immune saliva. Microneedle-based oral cavity vaccination was also compared to the intramuscular route using two HIV antigens, a virus-like particle and a DNA vaccine. Microneedle-based delivery to the oral cavity and the intramuscular route exhibited similar (p > 0.05) yet significant (p < 0.05) levels of antigen-specific IgG in serum. However, only the microneedle-based oral cavity vaccination group stimulated a significantly higher (p < 0.05) antigen-specific IgA response in saliva, but not intramuscular injection. CONCLUSION: In conclusion, this study provides a novel method using microneedles to induce systemic IgG and secretory IgA in saliva, and could offer a versatile technique for oral mucosal vaccination.

The impact of pregnancy on the HIV-1-specific T cell function in infected pregnant women.

Evidences indicate that pregnancy can alter the Ag-specific T-cell responses. This work aims to evaluate the impact of pregnancy on the in vitro HIV-1-specific immune response. As compared with non-pregnant patients, lower T-cell proliferation and higher IL-10 production were observed in T-cell cultures from pregnant patients following addition of either mitogens or HIV-1 antigens. In our system, the main T lymphocyte subset involved in producing IL-10 was CD4(+)FoxP3(-). Depletion of CD4(+) cells elevated TNF-α and IFN-γ production. Interestingly, the in vitro HIV-1 replication was lower in cell cultures from pregnant patients, and it was inversely related to IL-10 production. In these cultures, the neutralization of IL-10 by anti-IL-10 mAb elevated TNF-α release and HIV-1 replication. In conclusion, our results reveal that pregnancy-related events should favor the expansion of HIV-1-specific IL-10-secreting CD4(+) T-cells in HIV-1-infected women, which should, in the scenario of pregnancy, help to reduce the risk of vertical HIV-1 transmission.

Long peptides induce polyfunctional T cells against conserved regions of HIV-1 with superior breadth to single-gene vaccines in macaques.

A novel T-cell vaccine strategy designed to deal with the enormity of HIV-1 variation is described and tested for the first time in macaques to inform and complement approaching clinical trials. T-cell immunogen HIVconsv, which directs vaccine-induced responses to the most conserved regions of the HIV-1, proteome and thus both targets diverse clades in the population and reduces the chance of escape in infected individuals, was delivered using six different vaccine modalities: plasmid DNA (D), attenuated human (A) and chimpanzee (C) adenoviruses, modified vaccinia virus Ankara (M), synthetic long peptides, and Semliki Forest virus replicons. We confirmed that the initial DDDAM regimen, which mimics one of the clinical schedules (DDDCM), is highly immunogenic in macaques. Furthermore, adjuvanted synthetic long peptides divided into sub-pools and delivered into anatomically separate sites induced T-cell responses that were markedly broader than those elicited by traditional single-open-reading-frame genetic vaccines and increased by 30% the overall response magnitude compared with DDDAM. Thus, by improving both the HIV-1-derived immunogen and vector regimen/delivery, this approach could induce stronger, broader, and theoretically more protective T-cell responses than vaccines previously used in humans.

Protection of rhesus macaques against disease progression from pathogenic SHIV-89.6PD by vaccination with phage-displayed HIV-1 epitopes.

The antigenic polymorphism of HIV-1 is a major obstacle in developing an effective vaccine. Accordingly, we screened random peptide libraries (RPLs) displayed on phage with antibodies from HIV-infected individuals and identified an array of HIV-specific epitopes that behave as antigenic mimics of conformational epitopes of gp120 and gp41 proteins. We report that the selected epitopes are shared by a collection of HIV-1 isolates of clades A-F. The phage-borne epitopes are immunogenic in rhesus macaques, where they elicit envelope-specific antibody responses. Upon intravenous challenge with 60 MID50 of pathogenic SHIV-89.6PD, all monkeys became infected; however, in contrast to the naive and mock-immunized monkeys, four of five mimotope-immunized monkeys experienced lower levels of peak viremia, followed by viral set points of undetectable or transient levels of viremia and a mild decline of CD4+ T cells, and were protected from progression to AIDS-like illness. These results provide a new approach to the design of broadly protective HIV-1 vaccines.

Vaccines for the prevention of HIV-1 disease.

Clinical investigation in humans and experimental lentivirus infection in nonhuman primates have advanced our understanding of immune responses that control HIV-1 disease. Recently, immunization approaches in macaques have shown that the immune response can control viremia and improve clinical outcome. When such vaccine strategies are formulated to be similarly immunogenic in humans, they could form the basis for the development of candidate AIDS vaccines that would prevent infection, suppress progression to disease or reduce HIV-1 transmission in humans.

Rapid qualitative and quantitative analysis of T-cell responses in HIV-1-infected individuals receiving successful HAART and HIV-1 sero-negative controls: concomitant assessment of perforin, IFN-gamma and IL-4 secretion.

The enzyme-linked immunospot (ELIspot) assay is a highly sensitive and valuable tool for determining the frequency of cytokine-secreting T cells. It is essential to determine both frequencies and functional capabilities of antigen-specific T cells, including cytokine secretion, degranulation, and cytotoxicity in order to obtain a fuller picture of the immune status of an individual. We describe here for the first time a perforin-release ELIspot assay which, when used in combination with IFN-gamma and IL-4 ELIspots, permits rapid assessment of these functional parameters for antigen-specific T cells. Whole antigen or peptides from HIV-1, recall and other viral antigens were used for in vitro stimulation. Anti-HIV-1 responses in treated chronically infected individuals were weak, both in terms of perforin and IFN-gamma production. Tetanus toxoid stimulation was associated with moderate perforin release and a predominantly type-2 IL-4 producing response, whilst herpes simplex virus antigen stimulation resulted in perforin release but only a weak type-1 IFN-gamma response. Anti-cytomegalovirus responses generated high levels of perforin in conjunction with IFN-gamma. Cytokines IL-2 and IL-12/IL-15 induced perforin release coupled with an IFN-gamma type-1 response. Perforin release strongly correlated with IFN-gamma production to individual influenza, Epstein-Barr virus or cytomegalovirus MHC class I restricted peptides, in an HIV-1 sero-negative cohort, indicating a cytolytic type-1 CD8+ T-cell response. Evaluation of immunogenicity and putative efficacy of candidate vaccines using IFN-gamma will not be as informative alone as when combined with perforin and IL-4 evaluations, which allow assessment of specific cytotoxic potential without extensive cell culture.

Influence of aluminum-based adjuvant on the immune response to multiantigenic formulation.

Several adjuvants have been described and tested in humans. However, the aluminum-based adjuvants remain the most widely used component in vaccines today. Emerging data suggest that aluminum phosphate and aluminum hydroxide adjuvants do not promote a strong commitment to the helper T cell type 2 (Th2) pathway when they are coadministered with some Th1 adjuvants. In this regard, subtle differences between both aluminum-based adjuvants have been demonstrated. We have previously shown that subcutaneous immunization, in aluminum phosphate, of a mixture comprising the surface and core antigens of hepatitis B virus (HBV) and the multiepitopic protein CR3 of human immunodeficiency virus type 1 elicits a CR3-specific Th1 immune response. In these experiments, the antigens were adjuvated at the same time. As the final selection of the best adjuvant should be based on experimental evidence, we asked whether aluminum hydroxide allows a better Th1 immune deviation than aluminum phosphate. We also studied several ways to mix the antigens and the impact on CR3-specific interferon (IFN)-gamma secretion. Our findings indicate that aluminum hydroxide allows better Th1 immunodeviation than aluminum phosphate adjuvant for the mixture of HBV antigens and CR3. In addition, CR3-specific IFN-gamma secretion of the various formulations tested was the same irrespective of the order in which the antigens were combined.

Improving the sensitivity of the ELISPOT analyses of antigen-specific cellular immune responses in rhesus macaques.

The close similarities in hematopoietic and immune systems of humans and rhesus macaques (Macaca mulatta) make them the desired nonhuman primate animal model for developing vaccines against infectious diseases relevant to humans. The best example is the simian immunodeficiency virus infection in macaques as a model for AIDS, resulting from infection of humans by HIV-1. Vaccine efficacy against viruses depends on priming cell-mediated immunity by the use of sensitive assays that can accurately detect even small levels of antigen-specific T-cell responses that may otherwise be missed easily. With this in mind, we developed the dendritic cell enzyme-linked immunospot (DC-ELISPOT) protocol by incorporating antigen-pulsed DCs to stimulate lymphocytes, as opposed to the conventional ELISPOT assay, which cultures mixtures of various low-level populations of indigent antigen-presenting cells and responding lymphocytes with antigens. In rhesus macaques immunized with an HIV envelope peptide cocktail vaccine, the DC-ELISPOT protocol enabled more accurate enumeration of the cellular immune responses, as antigen-specific interferon-gamma-producing cells, with up to 18-fold increase in detection sensitivity compared with conventional ELISPOT and elimination of false negative results. The increased sensitivity of DC-ELISOT protocol is further validated in tests determining recall antigen-specific responses in human volunteers after tuberculin skin testing.

Characterization of humoral and cellular immune responses in mice induced by immunization with HIV-1 Nef regulatory protein encapsulated in poly(DL-lactide-co-glycolide) microparticles.

We have characterized the humoral and cellular immune responses of BALB/c mice immunized with HIV-1 Nef regulatory protein encapsulated in poly(DL-lactide-co-glycolide) PLG particles. Three groups of mice were immunized with Nef PLG, Nef in the presence of complete Freund's adjuvant (CFA) or Nef alone in PBS. When titers were compared 7 months after the last injection, anti-Nef titers in mice immunized with Nef PLG were still close to the maximum, whereas a significant decrease was observed in mice immunized with Nef alone (five times lower) or with Nef in CFA (three times lower). These results indicate that Nef PLG is at least a similar or better vector/adjuvant than Nef in CFA concerning the duration of the humoral immune response. The analysis of cytokine profiles (IL-5 and IL-10) and the isotypic patterns of anti-Nef antibodies (predominantly IgG1), in the three groups of mice, indicated a predominant Th2 immune response. Using synthetic peptides covering the entire sequence of Nef, we identified at least three linear epitopes within sequences 32-64, 118-167 and 185-205 in the sera of mice immunized with Nef PLG or Nef CFA. In contrast, anti-Nef antibodies against Nef alone failed to recognize synthetic peptides, indicating that the majority of anti-Nef antibodies were primarily directed against conformational epitopes. We then examined the ability of Nef PLG to prime for the antigen-specific proliferative responses in vitro. The data obtained indicate the presence of both B-cell and T-cell epitopes in the C-terminal fragment of the protein after immunization of mice with Nef encapsulated in PLG particles.

Angiogenic, lymphangiogenic and adipogenic effects of HIV-1 matrix protein p17.

Lymphangiogenesis and concurrent angiogenesis are essential in supporting proliferation and survival of AIDS-related lymphomas, which are often metastatic. In vitro studies suggest a candidate angiogienic and lymphangiogenic factor encoded by HIV: the matrix protein p17. p17 accumulates in lymph nodes of patients even when they are undergoing highly active antiretroviral therapy. p17 has been found to affect immune cells, and recent data showed that a variant p17, called S75X, induces cell growth by triggering MAPK/ERK and PI3K/AKT pathways. We tested the in vivo angiogenic activity of p17 by injecting it in Matrigel plugs in nude mice. Plugs were retrieved 7 days after injection, and assessed macroscopically, and by light and confocal microscopy. Our data revealed that both reference and S75X variant p17 promote angiogenesis and lymphangiogenesis in vivo. Our results suggest that the induction of angiogenesis and lymphangiogenesis by HIV-1 p17 may generate a favorable microenvironment that could trigger tumor growth and maintenance. Moreover, the presence of adipocytes infiltration observed at the histological level suggests a possible interplay between angiogenesis, lymphangiogenesis and adipogenesis. These findings offer new opportunities for the development of treatment strategies to combat HIV-related cancers.

HIV specific responses induced in nonhuman primates with ANRS HIV-Lipo-5 vaccine combined with rMVA-HIV prime or boost immunizations.

We evaluated the immunogenicity of a prime/boost vaccine strategy combining 5 lipopeptides (HIV-Lipo-5) and a recombinant modified vaccinia virus Ankara (rMVA-HIV) in cynomolgus macaques. Both of these vaccine components deliver HIV LAI Gag, Pol, and Nef antigens. Systemic and local safety was excellent in all groups. Immunization with HIV-Lipo-5 alone induced significant serum anti-HIV antibody titers which were not modified by rMVA-HIV immunization. However, induction of T-cell responses, as measured by IFNγ and IL-2 producing cells upon short-term stimulation with HIV peptide pools, required combined immunization with rMVA-HIV. Responses were preferentially observed against Gag antigen. Interestingly, HIV-Lipo-5 efficiently primed HIV induced T-cell responses upon the injection of rMVA-HIV, which may help to reduce the required number of vector injections. Our results provide a rationale for the use of a strategy involving HIV-Lipo-5 priming followed by rMVA-HIV booster immunization as a prophylactic or therapeutic vaccine approach against HIV infection and AIDS.

Antibody-dependent cellular cytotoxicity against HIV-1 in sera of immunized chimpanzees.

After immunization of chimpanzees against HIV antigens, antibodies that mediate antibody-dependent cellular cytotoxicity (ADCC) were evaluated and compared with anti-HIV-antibody levels detected by enzyme-linked immunosorbent assay (ELISA) and neutralizing antibody titers. Adult chimpanzees were immunized with different HIV-1 (LAV-BRU) antigen preparations: recombinant vaccinia virus (rVV) expressing gp160, p25 or p27nef; formalin- and beta-propiolactone-inactivated whole virus (inHIV); soluble recombinant gp160 either associated or not associated with other HIV proteins; a 25-mer peptide from the V3 region of gp120 coupled with KLH (V3-KLH). Immunization with the various rVV mixtures induced no or borderline ADCC increase above preimmune serum levels. Stronger and more sustained reactivity was elicited by inHIV. Purified HIV antigens elicited ADCC activity when the chimpanzees were naive; ADCC increased or remained at the same level when the animals had been preimmunized with rVV and/or inHIV. This type of reactivity apparently did not depend on whether gp160 alone or mixed with other proteins was used for immunization. The injection of V3-KLH resulted in only little, if any, recall ADCC response. ELISA antibody titers significantly correlated with ADCC and neutralizing antibody titers, but serum ADCC was independent of neutralizing antibody titers, an indication that the two latter serum activities are mediated by independent antibodies. Therefore, ADCC is elicited in the same manner as other antibody activities by the immunization of chimpanzees with inHIV or with purified recombinant HIV antigen preparations. The results obtained from the three chimpanzees of this series, which were subsequently challenged with infectious virus through the intravenous route, suggest that serum ADCC may be considered for vaccination purposes.

Safety and immunogenicity of multiple conventional immunizations administered during early HIV infection.

Twenty-one asymptomatic adults who had recently received multiple polysaccharide, live viral, and protein-derived vaccines were identified as being infected with human immunodeficiency virus (HIV). The mean subject age was 24 years (range 18-33); 20 of 21 (95%) were male. The mean T4 count was 523/mm3 with a mean T4/T8 ratio of 0.6. Serologic responses to immunization with meningococcus group C, adenovirus types 4 and 7, tetanus, and diphtheria were evaluated for the HIV seropositive subjects and were compared with the responses of similarly vaccinated age-, sex-, and race-matched HIV-seronegative controls. Significantly fewer (p less than 0.03) HIV subjects responded to meningococcus C (bactericidal antibody) and adenovirus 4 (neutralizing antibody) vaccines than did normals; the HIV-infected subjects who did respond produced functional antibody comparable to that of normals. Booster responses of HIV subjects to tetanus and diphtheria were comparable to those of normals. HIV-infected vaccine nonresponders did not differ from HIV-infected responders in total white blood cell, T4, T4/T8, total serum IgG, or delayed-type hypersensitivity skin test reactivity. All HIV subjects had negative cultures for live vaccine viruses (rubella, measles, adenovirus, and poliovirus). Postimmunization, no clinically apparent adverse reactions to vaccination were detected.

Monitoring HIV-specific CD8+ T cell responses by intracellular cytokine production.

Human immunodeficiency virus (HIV)-specific CD8+ T cells play an important role in controlling HIV infection. Accurate monitoring of these cells is crucial in determining the effects of HIV therapy and vaccine efficacy. Using an intracellular cytokine staining based assay, we are able to directly quantify functional HIV-specific CD8+ T cells. This assay is highly reproducible, and can be performed using both fresh and cryopreserved peripheral blood cells. Importantly, this assay can be used to examine multiple HIV-peptide epitopes simultaneously, and is independent of patient HLA haplotype. Here, we examine the HIV-specific CD8+ T cell response to 95 optimized HIV-derived cytotoxic T lymphocyte (CTL) epitopes in 21 HIV-infected patients of varying HLA haplotype, using peptide mixes and matrices. We find that when using mixes of multiple HIV peptides, the CD8+ T cell response to the mixture is equivalent to the sum of the responses to the individual peptides contained therein. Detailed comparison of the responses in patients suggests that most patients generate a diverse CD8+ T cell response, recognizing multiple HIV epitopes derived from HIV Gag, Pol, Env, or Nef. Although some patients sharing HLA alleles occasionally recognize common peptides, rarely are responses to those peptides dominant within the same group of patients. These results confirm our previous findings that the responses to single HIV-peptides are rarely representative of the entire HIV response.

Vaccination with CTL epitopes that escape: an alternative approach to HIV vaccine development?

This article describes a novel approach to HIV vaccine design that is, as yet, unproven and still in preliminary development. In rhesus macaques infected with simian immunodeficiency virus (SIV), we have identified particular cellular immune responses that select for viral variants during primary infection. We speculate that the detection of viral variants with altered amino acids in CTL epitopes implies the successful clearance of cells harboring wild-type virus. Here, we present our rationale suggesting why such potent early CTL responses that exert an antiviral effect may be particularly attractive targets for induction by candidate vaccines. Conventional wisdom suggests that regions of the virus that are structurally and functionally important will generally be well-conserved both among clades and within an infected host. Amino acid replacements within these well-conserved regions should be difficult for the virus to accommodate. Therefore, these regions are traditionally considered ideal targets for vaccine induced immune responses because they are refractory to CTL escape mutations. Many examples of these regions have been identified in both HIV-1 and SIV(mac) (J. Immunol. 162 (1999) 3727; J. Virol. 67 (1993) 438) and have been included in candidate vaccine formulations. Human clinical trials testing these vaccines are currently underway. Our proposed method of vaccination with CTL epitopes that escape explores an alternative hypothesis. Rather than engendering responses to regions of the virus that do not escape, we reason that vaccination needs to accelerate the development of the initial immune responses that effectively select for amino acid variants during acute infection. By examining CTL escape during the acute phase, we will identify CTL responses that the virus cannot tolerate and incorporate these responses into vaccines.

Characterization of immunostimulating complexes (ISCOMS) of HIV-1.

HIV-1, strain HTLV-III, propagated in H9 cells and purified by sucrose gradient centrifugation, was used as native antigen source for the preparation of immunostimulating complexes, HIV-iscoms. The major antigen detected in the iscom was the cell-derived HLA-DR, which readily could be removed from the virus lysate by immunosorbent. In the iscoms the HIV structural proteins MA p17, p55 and TM gp41 were identified; SU gp120 was present in only minute amounts in the virus lysate. The iscom particles appeared well preserved after freeze drying with a round shape, approximately 35 nm in diameter, comprising morphological subunits, assembled with icosahedral symmetry. Immunization experiments in mice reflected the antigen content of the iscoms. High antibody response was induced to HLA-DR in non-depleted iscoms. Major humoral responses were observed to the viral structural proteins MA p17, CA p24, p55, and also to TM gp41. A low or negligible antibody response to SU gp120 was induced by the HIV-iscoms. The negligible response was, however, overcome by the addition of recombinant gp160 to the virus lysate prior to formation of iscoms, resulting in a preparation evoking a clear serum antibody to gp160.