Autologous IgG antibodies block outgrowth of a substantial but variable fraction of viruses in the latent reservoir for HIV-1.

In untreated HIV-1 infection, rapid viral evolution allows escape from immune responses. Viral replication can be blocked by antiretroviral therapy. However, HIV-1 persists in a latent reservoir in resting CD4+ T cells, and rebound viremia occurs following treatment interruption. The reservoir, which is maintained in part by clonal expansion, can be measured using quantitative viral outgrowth assays (QVOAs) in which latency is reversed with T cell activation to allow viral outgrowth. Recent studies have shown that viruses detected in QVOAs prior to treatment interruption often differ from rebound viruses. We hypothesized that autologous neutralizing antibodies directed at the HIV-1 envelope (Env) protein might block outgrowth of some reservoir viruses. We modified the QVOA to reflect pressure from low concentrations of autologous antibodies and showed that outgrowth of a substantial but variable fraction of reservoir viruses is blocked by autologous contemporaneous immunoglobulin G (IgG). A reduction in outgrowth of >80% was seen in 6 of 15 individuals. This effect was due to direct neutralization. We established a phylogenetic relationship between rebound viruses and viruses growing out in vitro in the presence of autologous antibodies. Some large infected cell clones detected by QVOA carried neutralization-sensitive viruses, providing a cogent explanation for differences between rebound virus and viruses detected in standard QVOAs. Measurement of the frequency of reservoir viruses capable of outgrowth in the presence of autologous IgG might allow more accurate prediction of time to viral rebound. Ultimately, therapeutic immunization targeting the subset of variants resistant to autologous IgG might contribute to a functional cure.

Advances in HIV-1 Vaccine Development.

An efficacious HIV-1 vaccine is regarded as the best way to halt the ongoing HIV-1 epidemic. However, despite significant efforts to develop a safe and effective vaccine, the modestly protective RV144 trial remains the only efficacy trial to provide some level of protection against HIV-1 acquisition. This review will outline the history of HIV vaccine development, novel technologies being applied to HIV vaccinology and immunogen design, as well as the studies that are ongoing to advance our understanding of vaccine-induced immune correlates of protection.

Screening of primary gp120 immunogens to formulate the next generation polyvalent DNA prime-protein boost HIV-1 vaccines.

Our previous preclinical studies and a Phase I clinical trial DP6-001 have indicated that a polyvalent Env formulation was able to elicit broadly reactive antibody responses including low titer neutralizing antibody responses against viral isolates of subtypes A, B, C and AE. In the current report, a panel of 62 gp120 immunogens were screened in a rabbit model to identify gp120 immunogens that can elicit improved binding and neutralizing antibody responses and some of them can be included in the next polyvalent formulation. Only about 19% of gp120 immunogens in this panel were able to elicit neutralizing antibodies against greater than 50% of the viruses included in a high throughput PhenoSense neutralization assay when these immuongens were tested as a DNA prime followed by a fixed 5-valent gp120 protein vaccine boost. The new polyvalent formulation, using five gp120 immunogens selected from this subgroup, elicited improved quality of antibody responses in rabbits than the previous DP6-001 formulation. More significantly, this new polyvalent formulation elicited higher antibody responses against a panel of gp70V1/V2 antigens expressing V1/V2 sequences from diverse subtypes. Bioinformatics analysis supports the design of a 4-valent or 5-valent formulation using gp120 immunogens from this screening study to achieve a broad coverage against 16 HIV-1 subtypes.

Generation of human MHC (HLA-A11/DR1) transgenic mice for vaccine evaluation.

The rapid occurrence of emerging infectious diseases demonstrates an urgent need for a new preclinical experimental model that reliably replicates human immune responses. Here, a new homozygous humanized human leukocyte antigen (HLA)-A11/DR1 transgenic mouse (HLA-A11(+/+)/DR01(+/+)/H-2-ß2m(-/-)/IAß(-/-)) was generated by crossing HLA-A11 transgenic (Tg) mice with HLA-A2(+/+)/DR01(+/+)/H-2-ß2m(-/-)/IAß(-/-) mice. The HLA-A11-restricted immune response of this mouse model was then examined. HLA-A11 Tg mice expressing a chimeric major histocompatibility complex (MHC) molecule comprising the α1, α2, and ß2m domains of human HLA-A11 and the α3 transmembrane and cytoplasmic domains of murine H-2D(b) were generated. The correct integration of HLA-A11 and HLA-DR1 into the genome of the HLA-A11/DR1 Tg mice (which lacked the expression of endogenous H-2-I/II molecules) was then confirmed. Immunizing mice with a recombinant HBV vaccine or a recombinant HIV-1 protein resulted in the generation of IFN-γ-producing cytotoxic T lymphocyte (CTL) and antigen-specific antibodies. The HLA-A11-restricted CTL response was directed at HLA immunodominant epitopes. These mice represent a versatile animal model for studying the immunogenicity of HLA CTL epitopes in the absence of a murine MHC response. The established animal model will also be useful for evaluating and optimizing T cell-based vaccines and for studying differences in antigen processing between mice and humans.

Optimized and enhanced DNA plasmid vector based in vivo construction of a neutralizing anti-HIV-1 envelope glycoprotein Fab.

Monoclonal antibody preparations have demonstrated considerable clinical utility in the treatment of specific malignancies, as well as inflammatory and infectious diseases. Antibodies are conventionally delivered by passive administration, typically requiring costly large-scale laboratory development and production. Additional limitations include the necessity for repeat administrations, and the length of in vivo potency. Therefore, the development of methods to generate therapeutic antibodies and antibody like molecules in vivo, distinct from an active antigen-based immunization strategy, would have considerable clinical utility. In fact, adeno-associated viral (AAV) vector mediated delivery of immunoglobulin genes with subsequent generation of functional antibodies has recently been developed. As well, anon-viral vector mediated nucleic acid based delivery technology could permit the generation of therapeutic/prophylactic antibodies in vivo, obviating potential safety issues associated with viral vector based gene delivery. This delivery strategy has limitations as well, mainly due to very low in vivo production and expression of protein from the delivered gene. In the study reported here we have constructed an "enhanced and optimized" DNA plasmid technology to generate immunoglobulin heavy and light chains (i.e., Fab fragments) from an established neutralizing anti-HIV envelope glycoprotein monoclonal antibody (VRC01). This "enhanced" DNA (E-DNA) plasmid technology includes codon/RNA optimization, leader sequence utilization, as well as targeted potentiation of delivery and expression of the Fab immunoglobulin genes through use of "adaptive" in vivo electroporation. The results demonstrate that delivery by this method of a single administration of the optimized Fab expressing constructs resulted in generation of Fab molecules in mouse sera possessing high antigen specific binding and HIV neutralization activity for at least 7 d after injection, against diverse HIV isolates. Importantly, this delivery strategy resulted in a rapid increase (i.e., in as little as 48 h) in Fab levels when compared with protein-based immunization. The active generation of functional Fab molecules in vivo has important conceptual and practical advantages over conventional ex vivo generation, purification and passive delivery of biologically active antibodies. Further study of this technique for the rapid generation and delivery of immunoglobulin and immunoglobulin like molecules is highly relevant and timely.

High HIV-1 prevalence, risk behaviours, and willingness to participate in HIV vaccine trials in fishing communities on Lake Victoria, Uganda.

INTRODUCTION: HIV epidemics in sub-Saharan Africa are generalized, but high-risk subgroups exist within these epidemics. A recent study among fisher-folk communities (FFC) in Uganda showed high HIV prevalence (28.8%) and incidence (4.9/100 person-years). However, those findings may not reflect population-wide HIV rates in FFC since the study population was selected for high-risk behaviour. METHODS: Between September 2011 and March 2013, we conducted a community-based cohort study to determine the population representative HIV rates and willingness to participate (WTP) in hypothetical vaccine trials among FFC, Uganda. At baseline (September 2011-January 2012), a household enumeration census was done in eight fishing communities (one lakeshore and seven islands), after which a random sample of 2200 participants aged 18-49 years was selected from 5360 individuals. Interviewer-administered questionnaire data were collected on HIV risk behaviours and WTP, and venous blood was collected for HIV testing using rapid HIV tests with enzyme-linked immunosorbent assay (EIA) confirmation. Adjusted prevalence proportion ratios (adj.PPRs) of HIV prevalence were determined using log-binomial regression models. RESULTS: Overall baseline HIV prevalence was 26.7% and was higher in women than men (32.6% vs. 20.8%, p<0.0001). Prevalence was lower among fishermen (22.4%) than housewives (32.1%), farmers (33.1%) and bar/lodge/restaurant workers (37%). The adj.PPR of HIV was higher among women than men (adj.PPR =1.50, 95%; 1.20, 1.87) and participants aged 30-39 years (adj.PPR=1.40, 95%; 1.10, 1.79) and 40-49 years (adj.PPR=1.41, 95%; 1.04, 1.92) compared to those aged 18-24 years. Other factors associated with HIV prevalence included low education, previous marriage, polygamous marriage, alcohol and marijuana use before sex. WTP in hypothetical vaccine trials was 89.3% and was higher in men than women (91.2% vs. 87.3%, p=0.004) and among island communities compared to lakeshore ones (90.4% vs. 85.8%, p=0.004). CONCLUSIONS: The HIV prevalence in the general fisher-folk population in Uganda is similar to that observed in the "high-risk" fisher folk. FFC have very high levels of willingness to participate in future HIV vaccine trials.

Hyperimmune bovine colostrum as a low-cost, large-scale source of antibodies with broad neutralizing activity for HIV-1 envelope with potential use in microbicides.

Bovine colostrum (first milk) contains very high concentrations of IgG, and on average 1 kg (500 g/liter) of IgG can be harvested from each immunized cow immediately after calving. We used a modified vaccination strategy together with established production systems from the dairy food industry for the large-scale manufacture of broadly neutralizing HIV-1 IgG. This approach provides a low-cost mucosal HIV preventive agent potentially suitable for a topical microbicide. Four cows were vaccinated pre- and/or postconception with recombinant HIV-1 gp140 envelope (Env) oligomers of clade B or A, B, and C. Colostrum and purified colostrum IgG were assessed for cross-clade binding and neutralization against a panel of 27 Env-pseudotyped reporter viruses. Vaccination elicited high anti-gp140 IgG titers in serum and colostrum with reciprocal endpoint titers of up to 1 × 10(5). While nonimmune colostrum showed some intrinsic neutralizing activity, colostrum from 2 cows receiving a longer-duration vaccination regimen demonstrated broad HIV-1-neutralizing activity. Colostrum-purified polyclonal IgG retained gp140 reactivity and neutralization activity and blocked the binding of the b12 monoclonal antibody to gp140, showing specificity for the CD4 binding site. Colostrum-derived anti-HIV antibodies offer a cost-effective option for preparing the substantial quantities of broadly neutralizing antibodies that would be needed in a low-cost topical combination HIV-1 microbicide.

Interleukin-21 inhibits humoral response to an HIV DNA vaccine by enhancing Bcl-6 and Pax-5 expression.

Interleukin-21 (IL-21) is a T-cell-derived cytokine that modulates T-cell, B-cell, and natural killer cell responses. It is not known if it could be used as an adjuvant for HIV DNA vaccination. In our study, we investigated if a DNA construct expressing IL-21 (designated as pVAX-IL-21) as a molecule adjuvant could enhance antigen-specific immune responses to an HIV DNA vaccine (pGX-EnvC). We found that a higher level of antigen-specific cytotoxic responses was induced in BALB/C mice immunized with pGX-EnvC with the pVAX-IL-21 via electroporation. The increased response was associated with higher expression of IFN-γ in CD8⁺ T cells. In contrast, the administration of pVAX-IL-21 inhibited the antibody responses to HIV induced by the pGX-EnvC. The plasma cell inhibitory transcription factors B-cell lymphoma 6 protein (Bcl-6) and Pax-5 were increased in B cells from mice that had been immunized by HIV DNA vaccine plus pVAX-IL-21, suggesting that the expressed IL-21 may inhibit the differentiation from B cells to plasma cells. These results indicate that IL-21 could enhance CD8⁺ T-cell immunity, but inhibit humoral responses during HIV DNA vaccination.

Inadequate coordination of maternal and infant HIV services detrimentally affects early infant diagnosis outcomes in Lilongwe, Malawi.

OBJECTIVE: To assess the continuity of care and outcome of pediatric HIV prevention, testing, and treatment services, focusing on early infant diagnosis with DNA polymerase chain reaction (PCR). DESIGN: A retrospective observational cohort. METHODS: Maternal HIV antibody, infant HIV DNA PCR test results, and outcome data from HIV-infected infants from the prevention of mother-to-child transmission, early infant diagnosis, and pediatric HIV treatment programs operating in Lilongwe, Malawi, between 2004 and 2008 were collected, merged, and analyzed. RESULTS: Of the 14,669 pregnant women who tested HIV antibody positive, 7875 infants (53.7%) received HIV DNA PCR testing. One thousand eighty-four infants (13.8%) were HIV infected. Three hundred twenty (29.5%) children enrolled into pediatric HIV care, with 202 (63.1%) at the Baylor Center of Excellence. Among these, antiretroviral therapy was initiated on 110 infants (54.5%) whose median age was 9.1 months (interquartile range, 5.4-13.8) and a median of 2.5 months (interquartile range, 1.4-5.2) after HIV clinic registration. Sixty-nine HIV-infected infants (34.2%) died or were lost by December 2008. Initiation of antiretroviral therapy increased the likelihood of survival 7-fold (odds ratio, 7.1; 95% confidence interval, 3.68 to 13.70). CONCLUSIONS: Separate programs for maternal and infant HIV prevention and care services demonstrated high attrition rates of HIV-exposed and HIV-infected infants, elevated levels of mother-to-child transmission, late infant diagnosis, delayed pediatric antiretroviral therapy initiation, and high HIV-infected infant mortality. Antiretroviral therapy increased HIV-infected infant survival, emphasizing the urgent need for improved service coordination and strategies that increase access to infant HIV diagnosis, improve patient retention, and reduce antiretroviral therapy initiation delays.

A novel retinoic acid, catechin hydrate and mustard oil-based emulsion for enhanced cytokine and antibody responses against multiple strains of HIV-1 following mucosal and systemic vaccinations.

Non-replicating protein- or DNA-based antigens generally require immune-enhancing adjuvants and delivery systems. It has been particularly difficult to raise antibodies against gp120 of HIV-1, which constitutes an important approach in HIV vaccine design. While almost all effort in adjuvant research has focused on mimicking the pathogens and the danger signals they engender in the host, relatively little effort has been spent on nutritive approaches. In this study, a new nutritive immune-enhancing delivery system (NIDS) composed of vitamin A, a polyphenol-flavonoid, catechin hydrate, and mustard oil was tested for its adjuvant effect in immune responses against the gp120 protein of HIV-1(CN54). Following a combination of two mucosal and two systemic vaccinations of mice, we found significant enhancement of both local and systemic antibodies as well as cytokine responses. These data have important implications for vaccine and adjuvant design against HIV-1 and other pathogens.

Autologous blood donor screening indicated a lower prevalence of viral hepatitis in East vs West Germany: epidemiological benefit from established health resources.

Prevalence data concerning viral hepatitis and human immunodeficiency virus (HIV) in the general population are usually scarce. We aimed for a large cohort representative of the general population that required little funding. Autologous blood donors are relatively representative of the general population, and are tested for viral hepatitis and HIV in many countries. However, frequently these data are not captured for epidemiologic purposes. We analysed data from well over 35,000 autologous blood donors as recorded in 21 different transfusion centres for anti-hepatitis C virus (HCV), HBsAg and anti-HIV, as well as TPHA if available. We found a lower prevalence of hepatitis B virus and HCV in East vs West Germany, 0.2%vs 0.32% and 0.16%vs 0.32% respectively, which confirms earlier data in smaller cohorts, thus supporting the value of our approach. HIV was too rare to disclose significant differences, 0.01%vs 0.02%. TPHA was higher in East (0.34%) vs West Germany (0.29%) without significant differences. HCV was more frequent in women vs men. Transfusion institutes managing autologous blood donations should be used as a resource for epidemiological data relating to viral hepatitis and HIV, if such testing is performed routinely. This approach generates data relating to the general population with special emphasis on undiagnosed cases.

Serodeconversion of HIV antibody-positive AIDS patients following treatment with V-1 Immunitor.

It is extremely rare when HIV seropositive adult patients experience spontaneous loss of antibodies, that is, seroreversion. The disappearance of HIV antibodies was occasionally attributed to iatrogenic intervention-serodeconversion. Such interventions include: HAART; oral interferon; Chinese herbal remedies; and therapeutic AIDS vaccines derived from pooled blood. Oral therapeutic, alloimmune AIDS vaccine, V-1 Immunitor (V1), was administered to 60000 HIV-positive Thai patients. The administration of V1 resulted in serodeconversion among 23 individuals. The patient group consisted of 9 females (39%) and 14 males (61%) including two 2-year-old boys. The age range was 2-58 years with mean/median 29/29.3 years. Patients were tested seropositive for HIV at least once before being enrolled on V1. The duration of treatment until discovery of seronegative status ranged between 2 weeks and 15 months with average/median 7.2/8 months. Time to seronegativity was correlated with baseline disease stage (R = 0.62; P = .002). The seronegative status was positively associated with V1-induced undetectable or low viral load (R = 0.65; P = .0008). The odds ratio analysis comparing the outcome of our study with published surveys of diagnostic accuracy of laboratory tests suggested that the probability of HIV antibody testing error was remote (P < .000001). The possible causes responsible for this unusual phenomenon are discussed.

The effect of viral suppression on cross-sectional incidence testing in the johns hopkins hospital emergency department.

OBJECTIVE(S): : To determine the effect of viral suppression on cross-sectional incidence testing. METHODS: : In 2001 and 2003, patients entering the Johns Hopkins Hospital (JHH) Emergency Department (ED) were enrolled into an interview-based identity-unlinked serosurvey. All HIV-positive samples were tested by the Vironostika-less sensitive (LS) enzyme immunoassay (EIA) (Organon-Teknika, Charteston, SC) and an avidity assay to determine recent HIV infection. Additionally 16 samples from 8 previously characterized elite suppressors (ES) were tested by cross-sectional incidence assays. RESULTS: : HIV prevalence was 12% for the 2001 survey and 11% for the 2003 survey. Of the HIV-infected subjects, 18% did not know they were infected. The Vironostika-LS EIA determined that 6% (11 of 183) and 7% (17 of 243) of HIV-positive individuals in 2001and 2003, respectively, were recently infected. Avidity testing confirmed that 6 of 11 in 2001 and 5 of 17 in 2003 were newly infected, leaving 17 discrepant samples. All 17 discrepant samples were Western blot-positive and viral load undetectable, and 7 of 17 had antiretroviral drugs (ARVs) in their serum. Ten individuals were virally suppressed without ARVs and seemed incident by the Vironostika-LS EIA but chronic by avidity testing. These 10 subjects had similar testing profiles to the known 16 ES samples, because 9 of 16 were incident by the Vironostika-LS EIA and 0 of 16 were incident by avidity testing. CONCLUSIONS: : By removing the viral load-negative individuals and confirming the initial Vironostika-LS EIA results by avidity testing, the incidence estimate was lowered from 1.73% to 0.94% per year in 2001 and from 1.90% to 0.56% per year in 2003. Viral suppression affects the performance of the cross-sectional incidence tests, which rely on antibody titer. In addition, 2% (10 of 426) of all HIV-infected individuals who use the JHH ED for medical care seem to suppress HIV to undetectable levels without ARVs.

An adenovirus-based HIV subtype B prime/boost vaccine regimen elicits antibodies mediating broad antibody-dependent cellular cytotoxicity against non-subtype B HIV strains.

Although HIV subtype B predominates in North America and Western Europe, most HIV infections worldwide are non-subtype B. Globally effective AIDS vaccines need to elicit broad immunity against multiple HIV strains. In this study, 10 chimpanzees were intranasally primed sequentially with adenovirus type 5 (Ad5)- and Ad7-HIVMNenv/rev recombinants and boosted twice intramuscularly with heterologous oligomeric HIVSF162 gp140DeltaV2 protein in MF59 adjuvant. Sera were evaluated for binding, neutralizing, and antibody-dependent cellular cytotoxicity (ADCC) against HIV clades A, B, C, and CRF01_AE. The vaccine regimen elicited high-titered HIV subtype A, B, C and CRF01_AE gp120-binding antibodies. Sera from 7 of 10 vaccinated chimpanzees cross-neutralized the heterologous South African subtype C primary HIVTV-1 isolate. Significant cross-clade neutralization against other subtype A, C and E isolates was not observed. Sera from all animals mediated ADCC of cells coated with gp120 from HIV subtypes A and B. Nine of 10 animals also exhibited ADCC activity against HIV subtype C and CRF01_AE gp120-coated targets. This subtype B Ad-HIV recombinant prime/envelope protein boost regimen is a promising approach for eliciting broad ADCC activity against diverse HIV clades. Incorporating additional non-subtype B envelope genes and protein boosts in a multivalent strategy may be required to elicit broader neutralizing antibodies against non-subtype B HIV strains.

[Adaptation of different modifications of enzyme immunoassay and immune blot assay to assess the candidate vaccine HIVREPOL against HIV/AIDS].

A preclinical trial of the vaccine HIVREPOL provided a complex of methods for assessing the identity and specific activity of vaccines against HIVIAIDS. The identity of "HIVREPOL" has been assessed by indirect enzyme immunoassay (EIA): the vaccine specifically binds the antibodies of the sera from HIV-infected individuals. Immune blot assay was the most informative method for assessing the identity of the candidate vaccine. The sera from HIVREPOL-vaccinated mice recognized the proteins gp41, p24, p55 of cultured HIV1 on "New-Lay-Blot1" strips. The bands corresponding to p24 were revealed in the line blots "Blot-HIV-1/2+O" and "INNO-LIA-HIV-Confirmation". The specific activity of the HIVREPOL vaccine was confirmed from the reactivity of sera of the mice vaccinated with recombinant proteins of the immunosorbents available in EIA test systems for the detection of HIV antibodies. Competitive EIA established the antigen-binding activity of sera from HIVREPOL-vaccinated mice against the native reference HIV-1 antigen.

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.

Development of V2-deleted trimeric envelope vaccine candidates from human immunodeficiency virus type 1 (HIV-1) subtypes B and C.

The urgent need for a vaccine against HIV/AIDS requires that multiple strategies be employed and evaluated in a clinical setting. V2-loop deleted trimeric envelope (Env) immunogens (protein and DNA) from subtypes B and C human immunodeficiency virus type 1 (HIV-1) strains were produced for ongoing and future clinical evaluations with other HIV antigens, adjuvants and deliveries.

Evaluation of envelope vaccines derived from the South African subtype C human immunodeficiency virus type 1 TV1 strain.

Human immunodeficiency virus type 1 (HIV-1) subtype C infections are on the rise in Sub-Saharan Africa and Asia. Therefore, there is a need to develop an HIV vaccine capable of eliciting broadly reactive immune responses against members of this subtype. We show here that modified HIV envelope (env) DNA vaccines derived from the South African subtype C TV1 strain are able to prime for humoral responses in rabbits and rhesus macaques. Priming rabbits with DNA plasmids encoding V2-deleted TV1 gp140 (gp140TV1DeltaV2), followed by boosting with oligomeric protein (o-gp140TV1DeltaV2) in MF59 adjuvant, elicited higher titers of env-binding and autologous neutralizing antibodies than priming with DNA vaccines encoding the full-length TV1 env (gp160) or the intact TV1 gp140. Immunization with V2-deleted subtype B SF162 env and V2-deleted TV1 env together using a multivalent vaccine approach induced high titers of oligomeric env-binding antibodies and autologous neutralizing antibodies against both the subtypes B and C vaccine strains, HIV-1 SF162 and TV1, respectively. Low-level neutralizing activity against the heterologous South African subtype C TV2 strain, as well as a small subset of viruses in a panel of 13 heterologous primary isolates, was observed in some rabbits immunized with the V2-deleted vaccines. Immunization of rhesus macaques with the V2-deleted TV1 DNA prime/protein boost also elicited high titers of env-binding antibodies and moderate titers of autologous TV1 neutralizing antibodies. The pilot-scale production of the various TV1 DNA vaccine constructs and env proteins described here should provide an initial platform upon which to improve the immunogenicity of these subtype C HIV envelope vaccines.

High-dose recombinant Canarypox vaccine expressing HIV-1 protein, in seronegative human subjects.

BACKGROUND: In clinical trials, canarypox ALVAC-human immunodeficiency virus (HIV) vaccines have been shown to elicit human HIV-specific cytotoxic T lymphocyte (CTL) responses in some but not all healthy uninfected adults.Methods. A clinical trial was conducted to examine whether the vaccine vCP1452 would elicit a greater HIV-specific CTL response when given at a dose of 10(8.0) TCID50 (60 participants) than when given at the regular dose, 10(7.26) TCID50 (40 participants); as a control, a placebo vaccine preparation also was administered (10 participants). RESULTS: Two weeks after the last vaccination in a series, HIV-specific CTL responses were not significantly different when measured by either chromium-release assay (8% and 16% in the high- and regular-dose recipients, respectively) or interferon- gamma ELISpot assay (8% and 15% in the high- and regular-dose recipients, respectively); moreover, recipients of the higher dose had greater local and systemic reactions (P<.001). CONCLUSIONS: High reactogenicity associated with an increased dose of vCP1452 negates the need for further evaluation of this strategy to boost the frequency of HIV-specific CTL response in seronegative human subjects. Development of highly immunogenic canarypox vectors requires further work to optimize vector and insert design, as well as novel ways to increase dosage and to reduce reactogenicity.

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.