David A. Hokey

Combined effects of IL-12 and electroporation enhances the potency of DNA vaccination in macaques

DNA vaccines are a promising technology. Historically, however, the ability of DNA vaccines to induce high response rates and strong immune responses, especially antibody responses, in non-human primates and human clinical trials has proven suboptimal. Here, we performed a pilot study in rhesus macaques to evaluate whether we could improve the immunogenicity of DNA vaccines through the use of adjuvant technology and improved delivery systems. The study consisted of four groups of animals that received: DNA by intramuscular (IM) injection, DNA with plasmid-encoded IL-12 by IM injection, DNA by IM injection with in vivo electroporation (EP), and DNA with IL-12 by IM EP. Each group was immunized three times with optimized HIV gag and env constructs. Vaccine immunogenicity was assessed by IFNgamma ELISpot, CFSE proliferation, polyfunctional flow cytometry, and antibody ELISA. Similar to previous studies, use of IL-12 as an adjuvant increased the gag and env-specific cellular responses. The use of EP to enhance plasmid delivery resulted in dramatically higher cellular as well as humoral responses. Interestingly, the use of EP to administer the DNA and IL-12 adjuvant combination resulted in the induction of higher, more efficient responses such that a 10-fold increase in antigen-specific IFNgamma(+) cells compared to IM DNA immunization was observed after a single immunization. In addition to increases in the magnitude of IFNgamma production in the initial and memory responses, the combined approach resulted in enhancements in the proliferative capacity of antigen-specific CD8(+) T cells and the amount of polyfunctional cells capable of producing IL-2 and TNFalpha in addition to IFNgamma. These data suggest that adjuvant and improved delivery methods may be able to overcome previous immunogenicity limitations in DNA vaccine technology.

Protection against simian/human immunodeficiency virus (SHIV) 89.6P in macaques after coimmunization with SHIV antigen and IL-15 plasmid

The cell-mediated immune profile induced by a recombinant DNA vaccine was assessed in the simian/HIV (SHIV) and macaque model. The vaccine strategy included coimmunization of a DNA-based vaccine alone or in combination with an optimized plasmid encoding macaque IL-15 (pmacIL-15). We observed strong induction of vaccine-specific IFN-γ-producing CD8+ and CD4+ effector T cells in the vaccination groups. Animals were subsequently challenged with 89.6p. The vaccine groups were protected from ongoing infection, and the IL-15 covaccinated group showed a more rapidly controlled infection than the group treated with DNA vaccine alone. Lymphocytes isolated from the group covaccinated with pmacIL-15 had higher cellular proliferative responses than lymphocytes isolated from the macaques that received SHIV DNA alone. Vaccine antigen activation of lymphocytes was also studied for a series of immunological molecules. Although mRNA for IFN-γ was up-regulated after antigen stimulation, the inflammatory molecules IL-8 and MMP-9 were down-regulated. These observed immune profiles are potentially reflective of the ability of the different groups to control SHIV replication. This study demonstrates that an optimized IL-15 immune adjuvant delivered with a DNA vaccine can impact the cellular immune profile in nonhuman primates and lead to enhanced suppression of viral replication.

A novel liposomal adjuvant system, CAF01, promotes long-lived Mycobacterium tuberculosis-specific T-cell responses in human

Here, we report on a first-in-man trial where the tuberculosis (TB) vaccine Ag85B-ESAT-6 (H1) was adjuvanted with escalating doses of a novel liposome adjuvant CAF01. On their own, protein antigens cannot sufficiently induce immune responses in humans, and require the addition of an adjuvant system to ensure appropriate delivery and concomitant immune activation. To date no approved adjuvants are available for induction of cellular immunity, which seems essential for a number of vaccines, including vaccines against TB. We vaccinated four groups of human volunteers: a non-adjuvanted H1 group, followed by three groups with escalating doses of CAF01-adjuvanted H1 vaccine. All subjects were vaccinated at 0 and 8 weeks and followed up for 150 weeks. Vaccination did not cause local or systemic adverse effects besides transient soreness at the injection site. Two vaccinations elicited strong antigen-specific T-cell responses which persisted after 150 weeks follow-up, indicating the induction of a long-lasting memory response in the vaccine recipients. These results show that CAF01 is a safe and tolerable, Th1-inducing adjuvant for human TB vaccination trials and for vaccination studies in general where cellular immunity is required.

Induction of Antitumor Immunity In vivo Following Delivery of a Novel HPV-16 DNA Vaccine Encoding an E6/E7 Fusion antigen

Human papillomavirus type 16 (HPV-16) infection is associated with a majority of cervical cancers and a significant proportion of head and neck cancers. Here, we describe a novel-engineered DNA vaccine that encodes a HPV-16 consensus E6/E7 fusion gene (pConE6E7) with the goal of increasing its antitumor cellular immunity. Compared to an early stage HPV-16 E7 DNA vaccine (pE7), this construct was up to five times more potent in driving E7-specific cellular immune responses. Prophylactic administration of this vaccine resulted in 100% protection against HPV E6 and E7-expressing tumors. Therapeutic studies indicated that vaccination with pConE6E7 prevented or delayed the growth of tumors. Moreover, immunization with pConE6E7 could also partially overcome immune tolerance in E6/E7 transgenic mice. Such DNA immunogens are interesting candidates for further study to investigate mechanisms of tumor immune rejection in vivo.

Aerosol Vaccination with AERAS-402 Elicits Robust Cellular Immune Responses in the Lungs of Rhesus Macaques but Fails To Protect against High-Dose Mycobacterium tuberculosis Challenge

Development of a vaccine against pulmonary tuberculosis may require immunization strategies that induce a high frequency of Ag-specific CD4 and CD8 T cells in the lung. The nonhuman primate model is essential for testing such approaches because it has predictive value for how vaccines elicit responses in humans. In this study, we used an aerosol vaccination strategy to administer AERAS-402, a replication-defective recombinant adenovirus (rAd) type 35 expressing Mycobacterium tuberculosis Ags Ag85A, Ag85B, and TB10.4, in bacillus Calmette–Guérin (BCG)-primed or unprimed rhesus macaques. Immunization with BCG generated low purified protein derivative–specific CD4 T cell responses in blood and bronchoalveolar lavage. In contrast, aerosolized AERAS-402 alone or following BCG induced potent and stable Ag85A/b-specific CD4 and CD8 effector T cells in bronchoalveolar lavage that largely produced IFN-γ, as well as TNF and IL-2. Such responses induced by BCG, AERAS-402, or both failed to confer overall protection following challenge with 275 CFUs M. tuberculosis Erdman, although vaccine-induced responses associated with reduced pathology were observed in some animals. Anamnestic T cell responses to Ag85A/b were not detected in blood of immunized animals after challenge. Overall, our data suggest that a high M. tuberculosis challenge dose may be a critical factor in limiting vaccine efficacy in this model. However, the ability of aerosol rAd immunization to generate potent cellular immunity in the lung suggests that using different or more immunogens, alternative rAd serotypes with enhanced immunogenicity, and a physiological challenge dose may achieve protection against M. tuberculosis.

Activation drives PD-1 expression during vaccine-specific proliferation and following lentiviral infection in macaques

Recent data supports that increased expression of PD‐1, a negative regulator of immune function, is associated with T cell exhaustion during chronic viral infection. However, PD‐1 expression during acute infection and vaccination has not been studied in great detail in primates. Here, we examine PD‐1 expression on CD3+ T cells following DNA vaccination or lentiviral infection of macaques. Ex vivo peptide stimulation of PBMC from DNA‐vaccinated uninfected macaques revealed a temporal increase in PD‐1 expression in proliferating antigen‐specific CD8+ T cells. Following the initial increase, PD‐1 expression steadily declined as proliferation continued, with a concomitant increase in IFN‐γ secretion. Subsequent examination of PD‐1 expression on T cells from uninfected and lentivirus‐infected non‐vaccinated macaques revealed a significant increase in PD‐1 expression with lentiviral infection, consistent with previous reports. PD‐1 expression was highest on cells with activated memory and effector phenotypes. Despite their decreased telomere length, PD‐1hi T cell populations do not appear to have statistically significant uncapped telomeres, typically indicative of proliferative exhaustion, suggesting a different mechanistic regulation of proliferation by PD‐1. Our data indicate that PD‐1 expression is increased as a result of T cell activation during a primary immune response as well as during persistent immune activation in macaques.

The tuberculosis vaccine H4:IC31 is safe and induces a persistent polyfunctional CD4 T cell response in South African adults: A randomized controlled trial.

BACKGROUND New, more effective vaccines to prevent tuberculosis (TB) disease are needed urgently. H4:IC31 is an investigational vaccine that contains a fusion protein of the immunodominant antigens TB10.4 and Ag85B, formulated in IC31 adjuvant. We assessed the safety and immunogenicity of H4:IC31 in South African adults from a TB endemic setting. METHODS In this double blind, placebo controlled, phase I trial, Mycobacterium tuberculosis-uninfected, HIV-uninfected, healthy adults with a history of childhood BCG vaccination were randomly allocated to two intramuscular vaccinations with 5, 15, 50 or 150 μg H4 formulated in 500nmol IC31, two months apart. Vaccinees were followed for six months to assess safety; immunogenicity was measured by ELISpot and intracellular cytokine staining assays. RESULTS Thirty-two participants received H4:IC31 and 8 received placebo. Injection site adverse events were common but mild; mild fatigue was the most common systemic adverse event. Frequencies of adverse events did not differ between dosage groups. Detectable antigen-specific CD4 T cell responses were induced by all doses of H4:IC31, but doses below 50 μg induced the highest frequencies of CD4 T cells, comprised predominantly of IFN-γ(+)TNF-α(+)IL-2(+) or TNF-α(+)IL-2(+) cells. These memory responses persisted up to the end of follow up, on study day 182. CONCLUSIONS H4:IC31 demonstrated an acceptable safety profile and was immunogenic in South African adults. In this trial, the 15 μg dose appeared to induce the most optimal immune response.

Aerosol vaccines for tuberculosis: A fine line between protection and pathology

Pulmonary delivery of vaccines against airborne infection is being investigated worldwide, but there is limited effort directed at developing inhaled vaccines for tuberculosis (TB). This review addresses some of the challenges confronting vaccine development for TB and attempts to link these challenges to the promises of mucosal immunity offered by pulmonary delivery. There are several approaches working toward this goal including subunit vaccines, recombinant strains, a novel vaccine strain Mycobacterium w, and DNA vaccine approaches. While it is clear that lung-resident adaptive immunity is an attainable goal, vaccine platforms must ensure that damage to the lung is limited during both vaccination and when memory cells respond to pathogenic infection.

The novel tuberculosis vaccine, AERAS-402, is safe in healthy infants previously vaccinated with BCG, and induces dose-dependent CD4 and CD8T cell responses

BACKGROUND Efforts to reduce risk of tuberculosis disease in children include development of effective vaccines. Our aim was to test safety and immunogenicity of the new adenovirus 35-vectored tuberculosis vaccine candidate AERAS-402 in infants, administered as a boost following a prime with the Bacille Calmette-Guerin vaccine. METHODS In a phase 1 randomised, double-blind, placebo-controlled, dose-escalation trial, BCG-vaccinated infants aged 6-9 months were sequentially assigned to four study groups, then randomized to receive an increasing dose-strength of AERAS-402, or placebo. The highest dose group received a second dose of vaccine or placebo 56 days after the first. The primary study outcome was safety. Whole blood intracellular cytokine staining assessed immunogenicity. RESULTS Forty-two infants received AERAS-402 and 15 infants received placebo. During follow-up of 182 days, an acceptable safety profile was shown with no serious adverse events or discontinuations related to the vaccine. AERAS-402 induced a specific T cell response. A single dose of AERAS-402 induced CD4T cells predominantly expressing single IFN-γ whereas two doses induced CD4T cells predominantly expressing IFN-γ, TNF-α and IL-2 together. CD8T cells were induced and were more likely to be present after 2 doses of AERAS-402. CONCLUSIONS AERAS-402 was safe and immunogenic in healthy infants previously vaccinated with BCG at birth. Administration of the highest dose twice may be the most optimal vaccination strategy, based on the induced immunity. Multiple differences in T cell responses when infants are compared with adults vaccinated with AERAS-402, in the same setting and using the same whole blood intracellular cytokine assay, suggest specific strategies may be important for vaccination for each population.

A double-blind, randomised, placebo-controlled, dose-finding trial of the novel tuberculosis vaccine AERAS-402, an adenovirus-vectored fusion protein, in healthy, BCG-vaccinated infants

BACKGROUND Several novel tuberculosis vaccines are currently in clinical trials, including AERAS-402, an adenovector encoding a fusion protein of Mycobacterium tuberculosis antigens 85A, 85B, and TB10.4. A multicentred trial of AERAS-402 safety and immunogenicity in healthy infants was conducted in three countries in sub-Saharan Africa, using an adaptive design. METHODS In a double-blind, randomised, placebo-controlled, dose-finding trial, we enrolled BCG-vaccinated, HIV-uninfected infants aged 16-26 weeks. Infants in the safety/dose-finding phase received two doses of AERAS-402 across three dose levels, or placebo, intramuscularly on days 0 and 28. Infants in the expanded safety phase received three doses of the highest dose level, with the 3rd dose at day 280. Follow up for safety and immunogenicity was for up to two years. RESULTS We enrolled 206 infants (52 placebo and 154 AERAS-402 recipients) into the dose-finding phase and 281 (141 placebo and 140 AERAS-402 recipients) into the expanded safety phase. Safety data were acceptable across all dose levels. No vaccine-related deaths were recorded. A single serious adverse event of tachypnoea was deemed related to study vaccine. Antibodies directed largely against Ag85A and Ag85B were detected. Low magnitude CD4+ and CD8+ polyfunctional T cell responses were observed at all dose levels. The addition of a third dose of AERAS-402 at the highest dose level did not increase frequency or magnitude of antibody or CD8+ T cell responses. CONCLUSIONS AERAS-402 has an acceptable safety profile in infants and was well tolerated at all dose levels. Response rate was lower than previously seen in BCG vaccinated adults, and frequency and magnitude of antigen-specific T cells were not increased by a third dose of vaccine.