Relationships between the presence of anti-Tat antibody, DNA and RNA viral load

The possible relationships between the intensity of humoral response to full length Tat protein, the amount of proviral DNA reservoir in peripheral blood mononuclear cells and RNA viral load were analyzed in plasma samples obtained from a group of HIV-1 seropositive subjects, who never received any antiretroviral therapy. All HIV-1 patients showed detectable levels of serum IgG to full-length Tat by immunoenzymatic assay. We found a higher percentage of HIV-1 seropositive subjects with low levels of antibody in the presence of barely detectable proviral DNA copies (< or =10 copies/1.5x10(5) PBMCs) and a high anti-Tat antibody response accompanied by variable (from >10(1) to > or =10(3) copies/1.5x10(5) PBMCs) levels of DNA load (p=0.011). Moreover, an inverse relationship between anti-Tat antibody titers and HIV-1 RNA viral load was demonstrated HIV-1 seropositive patients. In HIV-1-infected patients, a strong humoral immune response against HIV-1 transactivating Tat protein, able to down-modulate viral replication in peripheral blood, does not seem to inhibit the number of proviral DNA molecules in peripheral blood mononuclear cells. Even though our data strongly confirm the "positive" role of anti-Tat antibody on viral replication, the persistence of significant amount of DNA viral load in peripheral blood mononuclear cells, despite high level of anti Tat antibody, suggests a more cautious approach to HIV-1 Tat-containing vaccines, able to stimulate an immune specific response to transactivating Tat protein sufficient in inhibiting circulating virus, but not completely efficient in decreasing proviral DNA integration.     

Biocompatible Anionic Polymeric Microspheres as Priming Delivery System for Effetive HIV/AIDS Tat-Based Vaccines

Here we describe a prime-boost regimen of vaccination in Macaca fascicularis that combines priming with novel anionic microspheres designed to deliver the biologically active HIV-1 Tat protein and boosting with Tat in Alum. This regimen of immunization modulated the IgG subclass profile and elicited a balanced Th1-Th2 type of humoral and cellular responses. Remarkably, following intravenous challenge with SHIV89.6P_cy243, vaccinees significantly blunted acute viremia, as compared to control monkeys, and this control was associated with significantly lower CD4⁺ T cell depletion rate during the acute phase of infection and higher ability to resume the CD4⁺ T cell counts in the post-acute and chronic phases of infection. The long lasting control of viremia was associated with the persistence of high titers anti-Tat antibodies whose profile clearly distinguished vaccinees in controllers and viremics. Controllers, as opposed to vaccinated and viremic cynos, exhibited significantly higher pre-challenge antibody responses to peptides spanning the glutamine-rich and the RGD-integrin-binding regions of Tat. Finally, among vaccinees, titers of anti-Tat IgG1, IgG3 and IgG4 subclasses had a significant association with control of viremia in the acute and post-acute phases of infection. Altogether these findings indicate that the Tat/H1D/Alum regimen of immunization holds promise for next generation vaccines with Tat protein or other proteins for which maintenance of the native conformation and activity are critical for optimal immunogenicity. Our results also provide novel information on the role of anti-Tat responses in the prevention of HIV pathogenesis and for the design of new vaccine candidates.     

Comparison of heterologous neutralizing antibody responses of human immunodeficiency virus type 1 (HIV-1)- and HIV-2-infected Senegalese patients: distinct patterns of breadth and magnitude distinguish HIV-1 and HIV-2 infections

Neutralizing antibody responses against heterologous isolates in human immunodeficiency virus type 1 (HIV-1) and HIV-2 infections were compared, and their relationships with established clinical markers of progression were examined. Neutralizing responses against 7 heterologous primary isolates and 1 laboratory strain were compared between 32 untreated HIV-1-infected subjects and 35 untreated HIV-2-infected subjects using a pseudotyped reporter virus assay. The breadth of the neutralizing response, defined as the proportion of panel viruses positively neutralized by patient plasma, was significantly greater among HIV-2-infected subjects than among HIV-1-infected subjects. Notably, for fully one-third of HIV-2 subjects, all viruses were effectively neutralized in our panel. Magnitudes of responses, defined as reciprocal 50% inhibitory concentration (IC(50)) titers for positive reactions, were significantly greater among HIV-1-infected subjects than among HIV-2-infected subjects. When plasma samples from HIV-1 patients were tested for cross-neutralization of HIV-2 and vice versa, we found that these intertype responses are very rare and their prevalences comparable in both HIV-1 and HIV-2 infection. The significantly higher magnitude of heterologous responses for HIV-1 compared to HIV-2 prompted us to examine associations with viremia, which is known to be significantly higher in HIV-1 infection. Importantly, there was a significant positive correlation between the IC(50) titer and viral load within both the HIV-1 and HIV-2 groups, suggesting heterologous antibodies may be driven by viral replication. We conclude that HIV-2 infection is characterized by a broad, low-magnitude intratype neutralization response, while HIV-1 is characterized by a narrower but higher-magnitude intratype response and that a significant positive association between the IC(50) titer and viremia is common to both HIV-1 and HIV-2 infections.     

T cell responses to human endogenous retroviruses in HIV-1 infection

Human endogenous retroviruses (HERVs) are remnants of ancient infectious agents that have integrated into the human genome. Under normal circumstances, HERVs are functionally defective or controlled by host factors. In HIV-1-infected individuals, intracellular defense mechanisms are compromised. We hypothesized that HIV-1 infection would remove or alter controls on HERV activity. Expression of HERV could potentially stimulate a T cell response to HERV antigens, and in regions of HIV-1/HERV similarity, these T cells could be cross-reactive. We determined that the levels of HERV production in HIV-1-positive individuals exceed those of HIV-1-negative controls. To investigate the impact of HERV activity on specific immunity, we examined T cell responses to HERV peptides in 29 HIV-1-positive and 13 HIV-1-negative study participants. We report T cell responses to peptides derived from regions of HERV detected by ELISPOT analysis in the HIV-1-positive study participants. We show an inverse correlation between anti-HERV T cell responses and HIV-1 plasma viral load. In HIV-1-positive individuals, we demonstrate that HERV-specific T cells are capable of killing cells presenting their cognate peptide. These data indicate that HIV-1 infection leads to HERV expression and stimulation of a HERV-specific CD8+ T cell response. HERV-specific CD8+ T cells have characteristics consistent with an important role in the response to HIV-1 infection: a phenotype similar to that of T cells responding to an effectively controlled virus (cytomegalovirus), an inverse correlation with HIV-1 plasma viral load, and the ability to lyse cells presenting their target peptide. These characteristics suggest that elicitation of anti-HERV-specific immune responses is a novel approach to immunotherapeutic vaccination. As endogenous retroviral sequences are fixed in the human genome, they provide a stable target, and HERV-specific T cells could recognize a cell infected by any HIV-1 viral variant. HERV-specific immunity is an important new avenue for investigation in HIV-1 pathogenesis and vaccine design.     

Differential regulation of the antibody responses to Gag and Env proteins of human immunodeficiency virus type 1.

We have studied the antibody responses to Env and Gag antigens of human immunodeficiency virus type 1 (HIV-1) in several cohorts of HIV-1-infected individuals: long-term nonprogressors, progressors to disease, acute seroconvertors, and recipients of HIV-1 protease inhibitors. We conclude that the antibody responses to Env and Gag antigens are differentially regulated and that changes in the plasma viral load in the measurable range (500 to 10(8) RNA copies per ml) do not directly affect the antibody responses to these HIV-1 proteins. We provide quantitative estimates of HIV-1-specific immunoglobulin G concentrations in plasma, which can be in excess of 1 mg/ml for both anti-gp120 and anti-p24 once the immune response to HIV-1 has stabilized after seroconversion. We discuss the apparent paradox that the absence of anti-Gag antibodies (which have, at best, limited antiviral activity) is indicative of disease progression, while the retention of anti-Env antibodies (which do have antiviral activity) is of limited (or no) prognostic value. We show that the disappearance of anti-Gag antibodies during disease progression is highly unlikely to be due to immune complexing; instead, we believe that it reflects the loss of T-cell help that is more necessary for the anti-Gag than the anti-Env response.     

Targeting Tat and IFN(alpha) as a therapeutic AIDS vaccine

Evolution to AIDS is characterized by a progressive cellular immune suppression. Although there is substantial evidence for several mechanisms involved in disrupting the immune response by induction of apoptosis in responder cells by contact with infected cells, we propose that humoral factors also play a role, and that one such factor is the extracellular form of the human immunodeficiency virus (HIV)-1 Tat protein and another is IFN(alpha). Both Tat and interferon-alpha (IFN(alpha)) inhibit antigen-stimulate T-cell proliferation, and specific anti-Tat and/or anti-IFN(alpha) Abs prevent generation of HIV-1-induced suppressor cells. We propose that high titer anti-Tat and/or anti-IFN(alpha) Abs, neutralizing extracellular Tat, and/or IFN(alpha), induced by vaccines described here, antagonize HIV-1-induced immunosuppression. Innocuous vaccines were prepared by using inactivated but immunogenic Tat (Toxoid) and inactivated and immunogenic IFN(alpha) (kinoid) derivatives. Both Tat Toxoid and IFN(alpha) kinoid were well tolerated and elicited specific neutralizing antibodies (Abs) in mice, monkeys, and seronegative and HIV-1-infected individuals.     

Saponin adjuvant enhancement of antigen-specific immune responses to an experimental HIV-1 vaccine.

The adjuvant activity of a single highly purified saponin from the soap bark tree Quillaja saponaria was evaluated by using it as a component in an experimental vaccine containing rHIV-1 envelope protein (HIV-1 160D) adsorbed to alum. BALB/c mice immunized with experimental vaccine formulations containing the saponin adjuvant QS-21 produced significantly higher titers of antibodies than mice vaccinated with only the alum-adsorbed HIV-1 160D. Potent amnestic antibody responses to HIV-1 viral proteins were also induced. Ag-specific proliferative responses to recombinant proteins and to three variants of HIV-1 were significantly increased using QS-21 as an adjuvant. Alum-adsorbed HIV-1 160D failed to induce measurable proliferative responses to inactivated HIV-1 viruses, but group-specific proliferative responses were raised when the QS-21 adjuvant was used in the vaccine formulation. MHC class I restricted CTL specific for the immunodominant V-3 loop were induced but only when the QS-21 adjuvant was included in the vaccine formulation. The production of serine esterase by Ag-activated splenic mononuclear cells, indicating the maturation of precursor CTL, was used as a secondary measure of CTL activity, and this response was also increased. The specificity of antibody responses was not significantly broadened using QS-21; the adjuvant increased the immune recognition of epitopes throughout the HIV-1 glycoprotein 160. However, the specificity of the proliferation and serine esterase responses was broadened, suggesting that the QS-21 augmented cell-mediated immune responses specific for epitopes outside of the V-3 loop. Additionally, the QS-21 adjuvant appeared to induce recognition of weakly immunogenic epitopes that were not recognized using only alum-adsorbed HIV-1 160D. The ability of QS-21 to augment both antibody and cell-mediated immune responses suggests that this adjuvant could be a valuable component in subunit vaccines.     

Initial B-cell responses to transmitted human immunodeficiency virus type 1: virion-binding immunoglobulin M (IgM) and IgG antibodies followed by plasma anti-gp41 antibodies with ineffective control of initial viremia

A window of opportunity for immune responses to extinguish human immunodeficiency virus type 1 (HIV-1) exists from the moment of transmission through establishment of the latent pool of HIV-1-infected cells. A critical time to study the initial immune responses to the transmitted/founder virus is the eclipse phase of HIV-1 infection (time from transmission to the first appearance of plasma virus), but, to date, this period has been logistically difficult to analyze. To probe B-cell responses immediately following HIV-1 transmission, we have determined envelope-specific antibody responses to autologous and consensus Envs in plasma donors from the United States for whom frequent plasma samples were available at time points immediately before, during, and after HIV-1 plasma viral load (VL) ramp-up in acute infection, and we have modeled the antibody effect on the kinetics of plasma viremia. The first detectable B-cell response was in the form of immune complexes 8 days after plasma virus detection, whereas the first free plasma anti-HIV-1 antibody was to gp41 and appeared 13 days after the appearance of plasma virus. In contrast, envelope gp120-specific antibodies were delayed an additional 14 days. Mathematical modeling of the earliest viral dynamics was performed to determine the impact of antibody on HIV replication in vivo as assessed by plasma VL. Including the initial anti-gp41 immunoglobulin G (IgG), IgM, or both responses in the model did not significantly impact the early dynamics of plasma VL. These results demonstrate that the first IgM and IgG antibodies induced by transmitted HIV-1 are capable of binding virions but have little impact on acute-phase viremia at the timing and magnitude that they occur in natural infection.     

Induction of neutralizing antibodies and Th1-polarized and CD4-independent CD8+ T-cell responses following delivery of human immunodeficiency virus type 1 Tat protein by recombinant adenylate cyclase of Bordetella pertussis

HIV-Tat, a conserved protein playing a key role in the early life cycle of the human immunodeficiency virus (HIV) has been proposed as a potential AIDS vaccine. An HIV-Tat-based vaccine should elicit a broad, long-lasting, and neutralizing immune response. We have previously demonstrated that the adenylate cyclase (CyaA) from Bordetella pertussis targets dendritic cells and delivers CD8(+) and CD4(+) T-cell epitopes into the major histocompatibility complex class I and class II presentation pathways. We have also showed that CyaA induced specific and protective cytotoxic T cell responses in vivo. Here, we designed a prototype vaccine based on the HIV type 1 Tat delivered by CyaA (CyaA-E5-Tat) and tested its capacity to induce HIV-Tat-specific cellular as well as antibody responses. We showed that immunization of mice by CyaA-E5-Tat in the absence of adjuvant elicited strong and long-lasting neutralizing anti-Tat antibody responses more efficient than those obtained after immunization with Tat toxoid in aluminum hydroxide adjuvant. Analyses of the anti-Tat immunoglobulin G isotypes and the cytokine pattern showed that CyaA-E5-Tat induced a Th1-polarized immune response in contrast to the Th2-polarized immune responses obtained with the Tat toxoid. In addition, our data demonstrated that HIV-Tat-specific gamma interferon-producing CD8(+) T cells were generated after vaccination with CyaA-E5-Tat in a CD4(+) T-cell-independent manner. Based on these findings, CyaA-E5-Tat represents an attractive vaccine candidate for both preventive and therapeutic vaccination involving CyaA as an efficient nonreplicative vector for protein delivery.     

Immunogenicity of HIV-1 IIIB and SHIV 89.6P Tat and Tat toxoids in rhesus macaques: induction of humoral and cellular immune responses

This study compared immune responses in rhesus macaques immunized with unmodified HIV-1 IIIB Tat, SHIV89.6P Tat, and carboxymethylated IIIB and 89.6P Tat toxoids. Immunization with either IIIB or 89.6P preparation induced high titer and broadly crossreactive serum anti-Tat IgG that recognized HIV-1 subtype-E and SIVmac251 Tat. However, the response was delayed, and titers were lower in 89.6P vaccination groups. Serum anti-Tat IgG recognized peptides corresponding to the amino-terminus, basic domain, and carboxy-terminal region. Cellular proliferative responses to Tat toxoids corresponding to the immunogen were evident in vitro in both IIIB and 89.6P groups. Crossreactive proliferative responses were observed in IIIB groups in response to stimulation with 89.6P or SIVmac251 Tat toxoids, but were much less prevalent in 89.6P groups. The truncated 86 amino acid IIIB Tat appears to be more immunogenic than the 102 amino acid 89.6P Tat with respect to both humoral and cellular immune responses, and may be a better vaccine component. Despite induction of robust humoral and cellular immune responses (including both CD4+ and CD8+ T-cell responses) to Tat, all animals were infected upon intravenous challenge with 30 MID(50) of SHIV89.6P and outcome of vaccine groups was not different from controls. Sequencing both Tat exons from serum viral RNA revealed no evidence of escape mutants. These results suggest that with intravenous SHIV89.6P challenge in rhesus macaques, precipitous CD4+ T-cell decline overwhelms potentially protective immune responses. Alternatively, Tat specific CD8+ T-cell responses may not appropriately recognize infected cells in vivo in this model. In view of evidence demonstrating Tat specific CTLs in the SIV model and in humans infected with HIV-1, results in this pathogenic SHIV model may not apparently predict the efficacy of this approach in human studies. The potency and cross-reactivity of these immune responses confirm Tat toxoid as an excellent candidate vaccine component.     

The Grafting of Universal T-Helper Epitopes Enhances Immunogenicity of HIV-1 Tat Concurrently Improving Its Safety Profile

Extracellular Tat (eTat) plays an important role in HIV-1 pathogenesis. The presence of anti-Tat antibodies is negatively correlated with disease progression, hence making Tat a potential vaccine candidate. The cytotoxicity and moderate immunogenicity of Tat however remain impediments for developing Tat-based vaccines. Here, we report a novel strategy to concurrently enhance the immunogenicity and safety profile of Tat. The grafting of universal helper T-lymphocyte (HTL) epitopes, Pan DR Epitope (PADRE) and Pol₇₁₁ into the cysteine rich domain (CRD) and the basic domain (BD) abolished the transactivation potential of the Tat protein. The HTL-Tat proteins elicited a significantly higher titer of antibodies as compared to the wild-type Tat in BALB/c mice. While the N-terminal epitope remained immunodominant in HTL-Tat immunizations, an additional epitope in exon-2 was recognized with comparable magnitude suggesting a broader immune recognition. Additionally, the HTL-Tat proteins induced cross-reactive antibodies of high avidity that efficiently neutralized exogenous Tat, thus blocking the activation of a Tat-defective provirus. With advantages such as presentation of multiple B-cell epitopes, enhanced antibody response and importantly, transactivation-deficient Tat protein, this approach has potential application for the generation of Tat-based HIV/AIDS vaccines.     

Inhibition of HIV-1 Tat-mediated LTR transactivation and HIV-1 infection by anti-Tat single chain intrabodies.

Genes encoding the rearranged immunoglobulin heavy and light chain variable regions of anti-HIV-1 Tat, exon 1 or exon 2 specific monoclonal antibodies have been used to construct single chain intracellular antibodies 'intrabodies' for expression in the cytoplasm of mammalian cells. These anti-Tat single chain intrabodies (anti-Tat sFvs) are additionally modified with a C-terminal human C kappa domain to increase cytoplasmic stability and/or the C-terminal SV40 nuclear localization signal to direct the nascent intrabody to the nuclear compartment, respectively. The anti-Tat sFvs with specific binding activity against the N-terminal activation domain of Tat, block Tat-mediated transactivation of HIV-1 LTR as well as intracellular trafficking of Tat in mammalian cells. As a result, the transformed lymphocytes expressing anti-Tat sFvs are resistant to HIV-1 infection. Thus, these studies demonstrate that stably expressed single chain intrabodies and their modified forms can effectively target molecules in the cytoplasm and nuclear compartments of eukaryotic cells. Furthermore, these studies suggest that anti-Tat sFvs used either alone or in combination with other genetically based strategies may be useful for the gene therapy of HIV-1 infection and AIDS.     

Generation of specific Th1 and CD8+ T-cell responses by immunization with mouse CD8+ dendritic cells loaded with HIV-1 viral lysate or envelope glycoproteins

Immunization with antigen-pulsed dendritic cells (DCs) can be used to elicit optimal immune responses. We developed the SRDC cell line, with a morphology, phenotype and activity similar to mouse splenic CD4(-)CD8alpha(+)CD205(+)CD11b(-) dendritic cells, which induce a polarized Th1 immune response. We evaluated the ability of SRDCs pulsed with HIV-1 viral lysate, oligomeric soluble gp140 or capsid p24 to induce specific antibody and T-cell responses in CBA/J mice. Immunization with all loaded SRDCs elicited antibody responses against the antigens tested. However, only HIV-1 viral lysate and gp140-pulsed SRDCs elicited specific CD4(+) and CD8(+) T-cell responses. These findings demonstrate the value of well characterized DC lines for optimizing the antigen-loading mixture, according to the DC population targeted. Our data suggest that splenic DCs pulsed with complex antigens, such as HIV-1 viral lysate or oligomeric soluble gp140, could be used as vaccines, eliciting strong primary Th1-polarized and humoral immune responses against HIV proteins in vivo.     

epitopes immediately below the base of the V3 loop of gp120 as targets for the initial autologous neutralizing antibody response in two HIV-1 subtype B-infected individuals

Epitopes that drive the initial autologous neutralizing antibody response in HIV-1-infected individuals could provide insights for vaccine design. Although highly strain specific, these epitopes are immunogenic, vulnerable to antibody attack on infectious virus, and could be involved in the ontogeny of broadly neutralizing antibody responses. To delineate such epitopes, we used site-directed mutagenesis, autologous plasma samples, and autologous monoclonal antibodies to map the amino acid changes that led to escape from the initial autologous neutralizing antibody response in two HIV-1 subtype B-infected individuals. Additional mapping of the epitopes was accomplished by using alanine scanning mutagenesis. Escape in the two individuals occurred by different pathways, but the responses in both cases appeared to be directed against the same region of gp120. In total, three amino acid positions were identified that were independently associated with autologous neutralization. Positions 295 and 332 are located immediately before and after the N- and C-terminal cysteines of the V3 loop, respectively, the latter of which affected an N-linked glycan that was critical to the neutralization epitope. Position 415 affected an N-linked glycan at position 413 in the C terminus of V4 that might mask epitopes near the base of V3. All three sites lie in close proximity on a four-stranded antiparallel sheet on the outer domain of gp120. We conclude that a region just below the base of the V3 loop, near the coreceptor binding domain of gp120, can be a target for autologous neutralization.     

The strength of the HIV-1 3' splice sites affects Rev function

Background

Of the diverse subtypes of Human Immunodeficiency Virus Type-1 (HIV-1), subtype-C strains cause a large majority of infections worldwide. The reasons for the global dominance of HIV-1 subtype-C infections are not completely understood. Tat, being critical for viral infectivity and pathogenesis, may differentially modulate pathogenic properties of the viral subtypes. Biochemical studies on Tat are hampered by the limitations of the current purification protocols. Tat purified using standard protocols often is competent for transactivation activity but defective for a variety of other biological functions. Keeping this limitation in view, we developed an efficient protein purification strategy for Tat.

Results

Tat proteins obtained using the novel strategy described here were free of contaminants and retained biological functions as evaluated in a range of assays including the induction of cytokines, upregulation of chemokine coreceptor, transactivation of the viral promoter and rescue of a Tat-defective virus. Given the highly unstable nature of Tat, we evaluated the effect of the storage conditions on the biological function of Tat following purification. Tat stored in a lyophilized form retained complete biological activity regardless of the storage temperature. To understand if variations in the primary structure of Tat could influence the secondary structure of the protein and consequently its biological functions, we determined the CD spectra of subtype-C and -B Tat proteins. We demonstrate that subtype-C Tat may have a relatively higher ordered structure and be less flexible than subtype-B Tat. We show that subtype-C Tat as a protein, but not as a DNA expression vector, was consistently inferior to subtype-B Tat in a variety of biological assays. Furthermore, using ELISA, we evaluated the anti-Tat antibody titers in a large number of primary clinical samples (n = 200) collected from all four southern Indian states. Our analysis of the Indian populations demonstrated that Tat is non-immunodominant and that a large variation exists in the antigen-specific antibody titers.

Conclusion

Our report not only describes a simple protein purification strategy for Tat but also demonstrates important structural and functional differences between subtype-B and -C Tat proteins. Furthermore, this is the first report of protein purification and characterization of subtype-C Tat.     

Effects of neutralizing antibodies on escape from CD8+ T-cell responses in HIV-1 infection

Despite substantial advances in our knowledge of immune responses against HIV-1 and of its evolution within the host, it remains unclear why control of the virus eventually breaks down. Here, we present a new theoretical framework for the infection dynamics of HIV-1 that combines antibody and CD8⁺ T-cell responses, notably taking into account their different lifespans. Several apparent paradoxes in HIV pathogenesis and genetics of host susceptibility can be reconciled within this framework by assigning a crucial role to antibody responses in the control of viraemia. We argue that, although escape from or progressive loss of quality of CD8⁺ T-cell responses can accelerate disease progression, the underlying cause of the breakdown of virus control is the loss of antibody induction due to depletion of CD4⁺ T cells. Furthermore, strong antibody responses can prevent CD8⁺ T-cell escape from occurring for an extended period, even in the presence of highly efficacious CD8⁺ T-cell responses.     

Heterologous epitope-scaffold prime:boosting immuno-focuses B cell responses to the HIV-1 gp41 2F5 neutralization determinant

The HIV-1 envelope glycoproteins (Env) gp120 and gp41 mediate entry and are the targets for neutralizing antibodies. Within gp41, a continuous epitope defined by the broadly neutralizing antibody 2F5, is one of the few conserved sites accessible to antibodies on the functional HIV Env spike. Recently, as an initial attempt at structure-guided design, we transplanted the 2F5 epitope onto several non-HIV acceptor scaffold proteins that we termed epitope scaffolds (ES). As immunogens, these ES proteins elicited antibodies with exquisite binding specificity matching that of the 2F5 antibody. These novel 2F5 epitope scaffolds presented us with the opportunity to test heterologous prime:boost immunization strategies to selectively boost antibody responses against the engrafted gp41 2F5 epitope. Such strategies might be employed to target conserved but poorly immunogenic sites on the HIV-1 Env, and, more generally, other structurally defined pathogen targets. Here, we assessed ES prime:boosting by measuring epitope specific serum antibody titers by ELISA and B cell responses by ELISpot analysis using both free 2F5 peptide and an unrelated ES protein as probes. We found that the heterologous ES prime:boosting immunization regimen elicits cross-reactive humoral responses to the structurally constrained 2F5 epitope target, and that incorporating a promiscuous T cell helper epitope in the immunogens resulted in higher antibody titers against the 2F5 graft, but did not result in virus neutralization. Interestingly, two epitope scaffolds (ES1 and ES2), which did not elicit a detectable 2F5 epitope-specific response on their own, boosted such responses when primed with the ES5. Together, these results indicate that heterologous ES prime:boost immunization regimens effectively focus the humoral immune response on the structurally defined and immunogen-conserved HIV-1 2F5 epitope.     

Purified envelope glycoproteins from human immunodeficiency virus type 1 variants induce individual, type-specific neutralizing antibodies.

Repeated immunizations of goats, horses, or chimpanzees with envelope glycoprotein gp120 isolated from human immunodeficiency virus type 1 (HIV-1) resulted in type-specific neutralizing-antibody responses, which began to decay approximately 20 days following the administration of antigen. This was true repeatedly for serum samples from animals hyperimmunized with gp120s from either the HTLV-IIIB (IIIB) or the envelope-divergent HTLV-IIIRF (RF) HIV-1 isolates. Animals previously immunized with the IIIB gp120 were then inoculated with purified RF gp120. The first response in these animals was an anamnestic resurgence of neutralizing antibody to IIIB without detectable neutralizing antibody for RF. However, with later RF gp120 boosts, the IIIB neutralizing-antibody titers fell and an RF type-specific neutralizing-antibody response developed. When assessed with other HIV-1 variants, no group-specific neutralizing antibody was seen in any of the vaccination protocols evaluated. These results will pose real obstacles in the development of an effective vaccine for HIV.