RV144 correlates of risk analysis showed that IgG antibodies to gp70V1V2 scaffolds inversely correlated with risk of HIV acquisition. We investigated IgG antibody responses in RV135 and RV132, two ALVAC-HIV prime-boost vaccine trials conducted in Thailand prior to RV144. Both trials used ALVAC-HIV (vCP1521) at 0, 1, 3, and 6 months and HIV-1 gp120MNgD and gp120A244gD in alum (RV135) or gp120SF2 and gp120CM235 in MF59 (RV132) at 3 and 6 months. We assessed ELISA binding antibodies to the envelope proteins (Env) 92TH023, A244gD and MNgD, cyclicV2, and gp70V1V2 CaseA2 (subtype B) and 92TH023 (subtype CRF01_AE), and Env-specific IgG1 and IgG3. Antibody responses to gp120 A244gD, MNgD, and gp70V1V2 92TH023 scaffold were significantly higher in RV135 than in RV132. Antibodies to gp70V1V2 CaseA2 were detected only in RV135 vaccine recipients and IgG1 and IgG3 antibody responses to A244gD were significantly higher in RV135. IgG binding to gp70V1V2 CaseA2 and CRF01_AE scaffolds was higher with the AIDSVAX®B/E boost but both trials showed similar rates of antibody decline post-vaccination. MF59 did not result in higher IgG antibody responses compared to alum with the antigens tested. However, notable differences in the structure of the recombinant proteins and dosage used for immunizations may have contributed to the magnitude and specificity of IgG induced by the two trials.
Background. The licensing of herpes zoster vaccine has demonstrated that therapeutic vaccination can help control chronic viral infection. Unfortunately, human trials of immunodeficiency virus (HIV) vaccine have shown only marginal efficacy.
Methods. In this double-blind study, 17 HIV-infected individuals with viral loads of <50 copies/mL and CD4+ T-cell counts of >350 cells/µL were randomly assigned to the vaccine or placebo arm. Vaccine recipients received 3 intramuscular injections of HIV DNA (4 mg) coding for clade B Gag, Pol, and Nef and clade A, B, and C Env, followed by a replication-deficient adenovirus type 5 boost (1010 particle units) encoding all DNA vaccine antigens except Nef. Humoral, total T-cell, and CD8+ cytotoxic T-lymphocyte (CTL) responses were studied before and after vaccination. Single-copy viral loads and frequencies of latently infected CD4+ T cells were determined.
Results. Vaccination was safe and well tolerated. Significantly stronger HIV-specific T-cell responses against Gag, Pol, and Env, with increased polyfunctionality and a broadened epitope-specific CTL repertoire, were observed after vaccination. No changes in single-copy viral load or the frequency of latent infection were observed.
Conclusions. Vaccination of individuals with existing HIV-specific immunity improved the magnitude, breadth, and polyfunctionality of HIV-specific memory T-cell responses but did not impact markers of viral control.
Clinical Trials Registration. NCT00270465
HIV; vaccination; therapy; cytotoxic T lymphocytes; humoral immunity; viral latency
In the RV144 HIV-1 vaccine efficacy trial, IgG antibody (Ab) binding levels to variable regions 1 and 2 (V1V2) of the HIV-1 envelope glycoprotein gp120 were an inverse correlate of risk of HIV-1 infection. To determine if V1V2-specific Abs cross-react with V1V2 from different HIV-1 subtypes, if the nature of the V1V2 antigen used to asses cross-reactivity influenced infection risk, and to identify immune assays for upcoming HIV-1 vaccine efficacy trials, new V1V2-scaffold antigens were designed and tested. Protein scaffold antigens carrying the V1V2 regions from HIV-1 subtypes A, B, C, D or CRF01_AE were assayed in pilot studies, and six were selected to assess cross-reactive Abs in the plasma from the original RV144 case-control cohort (41 infected vaccinees, 205 frequency-matched uninfected vaccinees, and 40 placebo recipients) using ELISA and a binding Ab multiplex assay. IgG levels to these antigens were assessed as correlates of risk in vaccine recipients using weighted logistic regression models. Levels of Abs reactive with subtype A, B, C and CRF01_AE V1V2-scaffold antigens were all significant inverse correlates of risk (p-values of 0.0008–0.05; estimated odds ratios of 0.53–0.68 per 1 standard deviation increase). Thus, levels of vaccine-induced IgG Abs recognizing V1V2 regions from multiple HIV-1 subtypes, and presented on different scaffolds, constitute inverse correlates of risk for HIV-1 infection in the RV144 vaccine trial. The V1V2 antigens provide a link between RV144 and upcoming HIV-1 vaccine trials, and identify reagents and methods for evaluating V1V2 Abs as possible correlates of protection against HIV-1 infection.
The Thai Phase III clinical trial (RV144) showed modest efficacy in preventing HIV-1 acquisition. Plasma collected from HIV-1-uninfected trial participants completing all injections with ALVAC-HIV (vCP1521) prime and AIDSVAX B/E boost were tested for antibody responses against HIV-1 gp120 envelope (Env). Peptide microarray analysis from six HIV-1 subtypes and group M consensus showed that vaccination induced antibody responses to the second variable (V2) loop of gp120 of multiple subtypes. We further evaluated V2 responses by ELISA and surface plasmon resonance using cyclic (Cyc) and linear V2 loop peptides. Thirty-one of 32 vaccine recipients tested (97%) had antibody responses against Cyc V2 at 2 weeks postimmunization with a reciprocal geometric mean titer (GMT) of 1100 (range: 200–3200). The frequency of detecting plasma V2 antibodies declined to 19% at 28 weeks post-last injection (GMT: 110, range: 100–200). Antibody responses targeted the mid-region of the V2 loop that contains conserved epitopes and has the amino acid sequence KQKVHALFYKLDIVPI (HXB2 Numbering sequence 169–184). Valine at position 172 was critical for antibody binding. The frequency of V3 responses at 2 weeks postimmunization was modest (18/32, 56%) with a GMT of 185 (range: 100–800). In contrast, naturally infected HIV-1 individuals had a lower frequency of antibody responses to V2 (10/20, 50%; p=0.003) and a higher frequency of responses to V3 (19/20, 95%), with GMTs of 400 (range: 100–3200) and 3570 (range: 200–12,800), respectively. RV144 vaccination induced antibodies that targeted a region of the V2 loop that contains conserved epitopes. Early HIV-1 transmission events involve V2 loop interactions, raising the possibility that anti-V2 antibodies in RV144 may have contributed to viral inhibition.
The RV144 clinical trial of a prime/boost immunizing regimen using recombinant canary pox (ALVAC-HIV) and two gp120 proteins (AIDSVAX B and E) was previously shown to have a 31.2% efficacy rate. Plasma specimens from vaccine and placebo recipients were used in an extensive set of assays to identify correlates of HIV-1 infection risk. Of six primary variables that were studied, only one displayed a significant inverse correlation with risk of infection: the antibody (Ab) response to a fusion protein containing the V1 and V2 regions of gp120 (gp70-V1V2). This finding prompted a thorough examination of the results generated with the complete panel of 13 assays measuring various V2 Abs in the stored plasma used in the initial pilot studies and those used in the subsequent case-control study. The studies revealed that the ALVAC-HIV/AIDSVAX vaccine induced V2-specific Abs that cross-react with multiple HIV-1 subgroups and recognize both conformational and linear epitopes. The conformational epitope was present on gp70-V1V2, while the predominant linear V2 epitope mapped to residues 165–178, immediately N-terminal to the putative α4β7 binding motif in the mid-loop region of V2. Odds ratios (ORs) were calculated to compare the risk of infection with data from 12 V2 assays, and in 11 of these, the ORs were ≤1, reaching statistical significance for two of the variables: Ab responses to gp70-V1V2 and to overlapping V2 linear peptides. It remains to be determined whether anti-V2 Ab responses were directly responsible for the reduced infection rate in RV144 and whether anti-V2 Abs will prove to be important with other candidate HIV vaccines that show efficacy, however, the results support continued dissection of Ab responses to the V2 region which may illuminate mechanisms of protection from HIV-1 infection and may facilitate the development of an effective HIV-1 vaccine.
In the RV144 trial, the estimated efficacy of a vaccine regimen against human immunodeficiency virus type 1 (HIV-1) was 31.2%. We performed a case–control analysis to identify antibody and cellular immune correlates of infection risk.
In pilot studies conducted with RV144 blood samples, 17 antibody or cellular assays met prespecified criteria, of which 6 were chosen for primary analysis to determine the roles of T-cell, IgG antibody, and IgA antibody responses in the modulation of infection risk. Assays were performed on samples from 41 vaccinees who became infected and 205 uninfected vaccinees, obtained 2 weeks after final immunization, to evaluate whether immune-response variables predicted HIV-1 infection through 42 months of follow-up.
Of six primary variables, two correlated significantly with infection risk: the binding of IgG antibodies to variable regions 1 and 2 (V1V2) of HIV-1 envelope proteins (Env) correlated inversely with the rate of HIV-1 infection (estimated odds ratio, 0.57 per 1-SD increase; P = 0.02; q = 0.08), and the binding of plasma IgA antibodies to Env correlated directly with the rate of infection (estimated odds ratio, 1.54 per 1-SD increase; P = 0.03; q = 0.08). Neither low levels of V1V2 antibodies nor high levels of Env-specific IgA antibodies were associated with higher rates of infection than were found in the placebo group. Secondary analyses suggested that Env-specific IgA antibodies may mitigate the effects of potentially protective antibodies.
This immune-correlates study generated the hypotheses that V1V2 antibodies may have contributed to protection against HIV-1 infection, whereas high levels of Env-specific IgA antibodies may have mitigated the effects of protective antibodies. Vaccines that are designed to induce higher levels of V1V2 antibodies and lower levels of Env-specific IgA antibodies than are induced by the RV144 vaccine may have improved efficacy against HIV-1 infection.
Background. A recombinant canarypox vector expressing human immunodeficiency virus type 1 (HIV-1) Gag, Pro, and membrane-linked gp120 (vCP1521), combined with a bivalent gp120 protein boost (AIDSVAX B/E), provided modest protection against HIV-1 infection in a community-based population in Thailand (RV144 trial). No protection was observed in Thai injection drug users who received AIDSVAX B/E alone (Vax003 trial). We compared the neutralizing antibody response in these 2 trials.
Methods. Neutralization was assessed with tier 1 and tier 2 strains of virus in TZM-bl and A3R5 cells.
Results. Neutralization of several tier 1 viruses was detected in both RV144 and Vax003. Peak titers were higher in Vax003 and waned rapidly in both trials. The response in RV144 was targeted in part to V3 of gp120.vCP1521 priming plus 2 boosts with gp120 protein was superior to 2 gp120 protein inoculations alone, confirming a priming effect for vCP1521. Sporadic weak neutralization of tier 2 viruses was detected only in Vax003 and A3R5 cells.
Conclusion. The results suggest either that weak neutralizing antibody responses can be partially protective against HIV-1 in low-risk heterosexual populations or that the modest efficacy seen in RV144 was mediated by other immune responses, either alone or in combination with neutralizing antibodies.
The severe acute respiratory syndrome (SARS) virus is a member of the Coronaviridae (CoV) family that first appeared in the Guangdong Province of China in 2002 and was recognized as an emerging infectious disease in March 2003. Over 8,000 cases and 900 deaths occurred during the epidemic. We report the safety and immunogenicity of a SARS DNA vaccine in a Phase I human study.
A single-plasmid DNA vaccine encoding the Spike (S) glycoprotein was evaluated in 10 healthy adults. Nine subjects completed the 3 dose vaccination schedule and were evaluated for vaccine safety and immune responses. Immune response was assessed by intracellular cytokine staining (ICS), ELISpot, ELISA, and neutralization assays.
The vaccine was well-tolerated. SARS-CoV-specific antibody was detected by ELISA in 8 of 10 subjects and neutralizing antibody was detected in all subjects who received 3 doses of vaccine. SARS-CoV-specific CD4+ T cell responses were detected in all vaccinees, and CD8+ T cell responses in ∼20% of individuals.
The VRC SARS DNA vaccine was well tolerated and produced cellular immune responses and neutralizing antibody in healthy adults.
T cell vaccine; emerging infectious disease; vaccine clinical trial
The Vaccine Research Center has developed vaccine candidates for different diseases/infectious agents (including HIV-1, Ebola, and Marburg viruses) built on an adenovirus vector platform, based on adenovirus type 5 or 35. To support clinical development of each vaccine candidate, pre-clinical studies were performed in rabbits to determine where in the body they biodistribute and how rapidly they clear, and to screen for potential toxicities (intrinsic and immunotoxicities). The vaccines biodistribute only to spleen, liver (Ad5 only), and/or iliac lymph node (Ad35 only) and otherwise remain in the site of injection muscle and overlying subcutis. Though ∼1011 viral particles were inoculated, already by Day 9, all but 103 to 105 genome copies per μg of DNA had cleared from the injection site muscle. By three months, the adenovector was cleared with, at most, a few animals retaining a small number of copies in the injection site, spleen (Ad5), or iliac lymph node (Ad35). This pattern of limited biodistribution and extensive clearance is consistent regardless of differences in adenovector type (Ad5 or 35), manufacturer's construct and production methods, or gene-insert. Repeated dose toxicology studies identified treatment-related toxicities confined primarily to the sites of injection, in certain clinical pathology parameters, and in body temperatures (Ad5 vectors) and food consumption immediately post-inoculation. Systemic reactogenicity and reactogenicity at the sites of injection demonstrated reversibility. These data demonstrate the safety and suitability for investigational human use of Ad5 or Ad35 adenovector-based vaccine candidates at doses of up to 2 × 1011 given intramuscularly to prevent various infectious diseases.
Adenovirus vector; Adenovirus Type 5; Adenovirus Type 35; HIV-1; Ebola; Marburg; Biodistribution; Repeated Dose Toxicology; Safety; Vaccine
West Nile virus (WNV) is a mosquitoborne flavivirus that can cause severe meningitis and encephalitis in infected individuals. We report the safety and immunogenicity of a WNV DNA vaccine in its first phase 1 human study.
A single-plasmid DNA vaccine encoding the premembrane and the envelope glycoproteins of the NY99 strain of WNV was evaluated in an open-label study in 15 healthy adults. Twelve subjects completed the 3-dose vaccination schedule, and all subjects completed 32 weeks of evaluation for safety and immunogenicity. The development of a vaccine-induced immune response was assessed by enzyme-linked immunosorbant assay, neutralization assays, intracelluar cytokine staining, and enzyme-linked immunospot assay.
The vaccine was safe and well tolerated, with no significant adverse events. Vaccine-induced T cell and antibody responses were detected in the majority of subjects. Neutralizing antibody to WNV was detected in all subjects who completed the 3-dose vaccination schedule, at levels shown to be protective in studies of horses, an incidental natural host for WNV.
Further assessment of this DNA platform for human immunization against WNV is warranted.
ClinicalTrials.gov identifier: NCT00106769.
Gene-based vaccine delivery is an important strategy in the development of a preventive vaccine for acquired immunodeficiency syndrome (AIDS). Vaccine Research Center (VRC) 004 is the first phase 1 dose-escalation study of a multiclade HIV-1 DNA vaccine.
VRC-HIVDNA009−00-VP is a 4-plasmid mixture encoding subtype B Gag-Pol-Nef fusion protein and modified envelope (Env) constructs from subtypes A, B, and C. Fifty healthy, uninfected adults were randomized to receive either placebo (n = 10) or study vaccine at 2 mg (n = 5), 4 mg (n = 20), or 8 mg (n = 15) by needle-free intramuscular injection. Humoral responses (measured by enzyme-linked immunosorbant assay, Western blotting, and neutralization assay) and T cell responses (measured by enzyme-linked immunospot assay and intracellular cytokine staining after stimulation with antigen-specific peptide pools) were measured.
The vaccine was well tolerated and induced cellular and humoral responses. The maximal CD4+ and CD8+ T cell responses occurred after 3 injections and were in response to Env peptide pools. The pattern of cytokine expression by vaccine-induced HIV-specific T cells evolved over time, with a diminished frequency of interferon-γ–producing T cells and an increased frequency of interleukin-2–producing T cells at 1 year.
DNA vaccination induced antibody to and T cell responses against 3 major HIV-1 subtypes and will be further evaluated as a potential component of a preventive AIDS vaccine regimen.
The development of an effective human immunodeficiency virus (HIV) vaccine is a high global priority. Here, we report the safety, tolerability, and immunogenicity of a replication-defective recombinant adenovirus serotype 5 (rAd5) vector HIV-1 candidate vaccine.
The vaccine is a mixture of 4 rAd5 vectors that express HIV-1 subtype B Gag-Pol fusion protein and envelope (Env) from subtypes A, B, and C. Healthy, uninfected adults were randomized to receive 1 intramuscular injection of placebo (n = 6) or vaccine at dose levels of 109 (n = 10), 1010 (n = 10), or 1011 (n = 10) particle units and were followed for 24 weeks to assess immunogenicity and safety.
The vaccine was well tolerated but was associated with more reactogenicity at the highest dose. At week 4, vaccine antigen–specific T cell responses were detected in 28 (93.3%) and 18 (60%) of 30 vaccine recipients for CD4+ and CD8+ T cells, respectively, by intracellular cytokine staining assay and in 22 (73%) of 30 vaccine recipients by enzyme-linked immunospot assay. Env-specific antibody responses were detected in 15 (50%) of 30 vaccine recipients by enzyme-linked immunosorbant assay and in 28 (93.3%) of 30 vaccine recipients by immunoprecipitation followed by Western blotting. No neutralizing antibody was detected.
A single injection induced HIV-1 antigen–specific CD4+ T cell, CD8+ T cell, and antibody responses in the majority of vaccine recipients. This multiclade rAd5 HIV-1 vaccine is now being evaluated in combination with a multiclade HIV-1 DNA plasmid vaccine.
The Vaccine Research Center has developed a number of vaccine candidates for different diseases/infectious agents (HIV-1, Severe Acute Respiratory Syndrome virus, West Nile virus, and Ebola virus, plus a plasmid cytokine adjuvant—IL-2/Ig) based on a DNA plasmid vaccine platform. To support the clinical development of each of these vaccine candidates, preclinical studies were performed to screen for potential toxicities (intrinsic and immunotoxicities). All treatment-related toxicities identified in these repeated-dose toxicology studies have been confined primarily to the sites of injection and seem to be the result of both the delivery method (as they are seen in both control and treated animals) and the intended immune response to the vaccine (as they occur with greater frequency and severity in treated animals). Reactogenicity at the site of injection is generally seen to be reversible as the frequency and severity diminished between doses and between the immediate and recovery termination time points. This observation also correlated with the biodistribution data reported in the companion article (Sheets et al., 2006), in which DNA plasmid vaccine was shown to remain at the site of injection, rather than biodistributing widely, and to clear over time. The results of these safety studies have been submitted to the Food and Drug Administration to support the safety of initiating clinical studies with these and related DNA plasmid vaccines. Thus far, standard repeated-dose toxicology studies have not identified any target organs for toxicity (other than the injection site) for our DNA plasmid vaccines at doses up to 8 mg per immunization, regardless of disease indication (i.e., expressed gene-insert) and despite differences (strengths) in the promoters used to drive this expression. As clinical data accumulate with these products, it will be possible to retrospectively compare the safety profiles of the products in the clinic to the results of the repeated-dose toxicology studies, in order to determine the utility of such toxicology studies for signaling potential immunotoxicities or intrinsic toxicities from DNA vaccines. These data build on the biodistribution studies performed (see companion article, Sheets et al., 2006) to demonstrate the safety and suitability for investigational human use of DNA plasmid vaccine candidates for a variety of infectious disease prevention indications.
DNA vaccines; HIV/AIDS; SARS; WNV; Ebola; DNA vaccine toxicology; plasmid vaccines
The Vaccine Research Center has developed a number of vaccine candidates for different diseases/infectious agents (HIV-1, Severe Acute Respiratory Syndrome virus, West Nile virus, and Ebola virus, plus a plasmid cytokine adjuvant—IL-2/Ig) based on a DNA plasmid vaccine platform. To support the clinical development of each of these vaccine candidates, preclinical studies have been performed in mice or rabbits to determine where in the body these plasmid vaccines would biodistribute and how rapidly they would clear. In the course of these studies, it has been observed that regardless of the gene insert (expressing the vaccine immunogen or cytokine adjuvant) and regardless of the promoter used to drive expression of the gene insert in the plasmid backbone, the plasmid vaccines do not biodistribute widely and remain essentially in the site of injection, in the muscle and overlying subcutis. Even though ∼ 1014 molecules are inoculated in the studies in rabbits, by day 8 or 9(∼ 1 week postinoculation), already all but on the order of 104−106 molecules per microgram of DNA extracted from tissue have been cleared at the injection site. Over the course of 2 months, the plasmid clears from the site of injection with only a small percentage of animals (generally 10−20%) retaining a small number of copies (generally around 100 copies) in the muscle at the injection site. This pattern of biodistribution (confined to the injection site) and clearance (within 2 months) is consistent regardless of differences in the promoter in the plasmid backbone or differences in the gene insert being expressed by the plasmid vaccine. In addition, integration has not been observed with plasmid vaccine candidates inoculated i.m. by Biojector 2000 or by needle and syringe. These data build on the repeated-dose toxicology studies performed (see companion article, Sheets et al., 2006) to demonstrate the safety and suitability for investigational human use of DNA plasmid vaccine candidates for a variety of infectious disease prevention indications.
DNA vaccines; HIV/AIDS; SARS; WNV; Ebola; DNA vaccine biodistribution; DNA vaccine integration; plasmid vaccines
To evaluate the safety and immunogenicity of a candidate HIV DNA vaccine administered using a needle-free device.
In this phase 1, dose escalation, double-blind, placebo-controlled clinical trial, 21 healthy adults were randomized to receive placebo or 0.5, 1.5, or 4 mg of a single plasmid expressing a Gag/Pol fusion protein. Each participant received repeat immunizations at days 28 and 56 after the first inoculation. Safety and immunogenicity data were collected.
The vaccine was well tolerated, with most adverse events being mild injection site reactions, including pain, tenderness, and erythema. No dose-limiting toxicities occurred. HIV-specific antibody response was not detected in any vaccinee by enzyme-linked immunosorbent assay. HIV-specific T-cell responses to Gag or Pol as measured by enzyme-linked immunospot assay and intracellular cytokine staining were of low frequency and magnitude.
This candidate HIV DNA vaccine was safe and well tolerated. No HIV-specific antibody responses were detected, and only low-magnitude HIV-specific T-cell responses were detected in 8 (53%) of 15 vaccinees. This initial product led to the development of a 4-plasmid multiclade HIV DNA Vaccine Research Center vaccine candidate in which envelope genes expressing Env from clades A, B, and C and a Nef gene from clade B have been added.
CD4+ T-cell immune response; gene delivery; immunization; needle-free device; plasmid vaccine; safety
Ebola viruses represent a class of filoviruses that causes severe hemorrhagic fever with high mortality. Recognized first in 1976 in the Democratic Republic of Congo, outbreaks continue to occur in equatorial Africa. A safe and effective Ebola virus vaccine is needed because of its continued emergence and its potential for use for biodefense. We report the safety and immunogenicity of an Ebola virus vaccine in its first phase I human study. A three-plasmid DNA vaccine encoding the envelope glycoproteins (GP) from the Zaire and Sudan/Gulu species as well as the nucleoprotein was evaluated in a randomized, placebo-controlled, double-blinded, dose escalation study. Healthy adults, ages 18 to 44 years, were randomized to receive three injections of vaccine at 2 mg (n = 5), 4 mg (n = 8), or 8 mg (n = 8) or placebo (n = 6). Immunogenicity was assessed by enzyme-linked immunosorbent assay (ELISA), immunoprecipitation-Western blotting, intracellular cytokine staining (ICS), and enzyme-linked immunospot assay. The vaccine was well-tolerated, with no significant adverse events or coagulation abnormalities. Specific antibody responses to at least one of the three antigens encoded by the vaccine as assessed by ELISA and CD4+ T-cell GP-specific responses as assessed by ICS were detected in 20/20 vaccinees. CD8+ T-cell GP-specific responses were detected by ICS assay in 6/20 vaccinees. This Ebola virus DNA vaccine was safe and immunogenic in humans. Further assessment of the DNA platform alone and in combination with replication-defective adenoviral vector vaccines, in concert with challenge and immune data from nonhuman primates, will facilitate evaluation and potential licensure of an Ebola virus vaccine under the Animal Rule.