Pre-Clinical Development of a Recombinant, Replication-Competent Adenovirus Serotype 4 Vector Vaccine Expressing HIV-1 Envelope 1086 Clade C

BackgroundThere is a well-acknowledged need for an effective AIDS vaccine that protects against HIV-1 infection or limits in vivo viral replication. The objective of these studies is to develop a replication-competent, vaccine vector based on the adenovirus serotype 4 (Ad4) virus expressing HIV-1 envelope (Env) 1086 clade C glycoprotein. Ad4 recombinant vectors expressing Env gp160 (Ad4Env160), Env gp140 (Ad4Env140), and Env gp120 (Ad4Env120) were evaluated.MethodsThe recombinant Ad4 vectors were generated with a full deletion of the E3 region of Ad4 to accommodate the env gene sequences. The vaccine candidates were assessed in vitro following infection of A549 cells for Env-specific protein expression and for posttranslational transport to the cell surface as monitored by the binding of broadly neutralizing antibodies (bNAbs). The capacity of the Ad4Env vaccines to induce humoral immunity was evaluated in rabbits for Env gp140 and V1V2-specific binding antibodies, and HIV-1 pseudovirus neutralization. Mice immunized with the Ad4Env160 vaccine were assessed for IFNγ T cell responses specific for overlapping Env peptide sets.ResultsRobust Env protein expression was confirmed by western blot analysis and recognition of cell surface Env gp160 by multiple bNAbs. Ad4Env vaccines induced humoral immune responses in rabbits that recognized Env 1086 gp140 and V1V2 polypeptide sequences derived from 1086 clade C, A244 clade AE, and gp70 V1V2 CASE A2 clade B fusion protein. The immune sera efficiently neutralized tier 1 clade C pseudovirus MW965.26 and neutralized the homologous and heterologous tier 2 pseudoviruses to a lesser extent. Env-specific T cell responses were also induced in mice following Ad4Env160 vector immunization.ConclusionsThe Ad4Env vaccine vectors express high levels of Env glycoprotein and induce both Env-specific humoral and cellular immunity thus supporting further development of this new Ad4 HIV-1 Env vaccine platform in Phase 1 clinical trials

Pre-Clinical Evaluation of a Replication-Competent Recombinant Adenovirus Serotype 4 Vaccine Expressing Influenza H5 Hemagglutinin

Influenza virus remains a significant health and social concern in part because of newly emerging strains, such as avian H5N1 virus. We have developed a prototype H5N1 vaccine using a recombinant, replication-competent Adenovirus serotype 4 (Ad4) vector, derived from the U.S. military Ad4 vaccine strain, to express the hemagglutinin (HA) gene from A/Vietnam/1194/2004 influenza virus (Ad4-H5-Vtn). Our hypothesis is that a mucosally-delivered replicating Ad4-H5-Vtn recombinant vector will be safe and induce protective immunity against H5N1 influenza virus infection and disease pathogenesis.The Ad4-H5-Vtn vaccine was designed with a partial deletion of the E3 region of Ad4 to accommodate the influenza HA gene. Replication and growth kinetics of the vaccine virus in multiple human cell lines indicated that the vaccine virus is attenuated relative to the wild type virus. Expression of the HA transgene in infected cells was documented by flow cytometry, western blot analysis and induction of HA-specific antibody and cellular immune responses in mice. Of particular note, mice immunized intranasally with the Ad4-H5-Vtn vaccine were protected against lethal H5N1 reassortant viral challenge even in the presence of pre-existing immunity to the Ad4 wild type virus.Several non-clinical attributes of this vaccine including safety, induction of HA-specific humoral and cellular immunity, and efficacy were demonstrated using an animal model to support Phase 1 clinical trial evaluation of this new vaccine

Passive Transfer of Immune Sera Induced by a Zika Virus-Like Particle Vaccine Protects AG129 Mice Against Lethal Zika Virus Challenge

Zika virus (ZIKV) poses a serious public health threat due to its association with birth defects in developing fetuses and Guillain-Barré Syndrome in adults. We are developing a ZIKV vaccine based on virus-like particles (VLPs) generated in transiently transfected HEK293 cells. The genetic construct consists of the prM and envelope structural protein genes of ZIKV placed downstream from a heterologous signal sequence. To better understand the humoral responses and correlates of protection (CoP) induced by the VLP vaccine, we evaluated VLP immunogenicity with and without alum in immune-competent mice (C57Bl/6 x Balb/c) and observed efficient induction of neutralizing antibody as well as a dose-sparing effect of alum. To assess the efficacy of the immune sera, we performed passive transfer experiments in AG129 mice. Mice that received the immune sera prior to ZIKV infection demonstrated significantly reduced viral replication as measured by viral RNA levels in the blood and remained healthy, whereas control mice succumbed to infection. The results underscore the protective effect of the antibody responses elicited by this ZIKV VLP vaccine candidate. These studies will help define optimal vaccine formulations, contribute to translational efforts in developing a vaccine for clinical development, and assist in the definition of immunologic CoP

Pre-clinical development of a recombinant, replication-competent adenovirus serotype 4 vector vaccine expressing HIV-1 envelope 1086 clade C.

There is a well-acknowledged need for an effective AIDS vaccine that protects against HIV-1 infection or limits in vivo viral replication. The objective of these studies is to develop a replication-competent, vaccine vector based on the adenovirus serotype 4 (Ad4) virus expressing HIV-1 envelope (Env) 1086 clade C glycoprotein. Ad4 recombinant vectors expressing Env gp160 (Ad4Env160), Env gp140 (Ad4Env140), and Env gp120 (Ad4Env120) were evaluated.The recombinant Ad4 vectors were generated with a full deletion of the E3 region of Ad4 to accommodate the env gene sequences. The vaccine candidates were assessed in vitro following infection of A549 cells for Env-specific protein expression and for posttranslational transport to the cell surface as monitored by the binding of broadly neutralizing antibodies (bNAbs). The capacity of the Ad4Env vaccines to induce humoral immunity was evaluated in rabbits for Env gp140 and V1V2-specific binding antibodies, and HIV-1 pseudovirus neutralization. Mice immunized with the Ad4Env160 vaccine were assessed for IFNγ T cell responses specific for overlapping Env peptide sets.Robust Env protein expression was confirmed by western blot analysis and recognition of cell surface Env gp160 by multiple bNAbs. Ad4Env vaccines induced humoral immune responses in rabbits that recognized Env 1086 gp140 and V1V2 polypeptide sequences derived from 1086 clade C, A244 clade AE, and gp70 V1V2 CASE A2 clade B fusion protein. The immune sera efficiently neutralized tier 1 clade C pseudovirus MW965.26 and neutralized the homologous and heterologous tier 2 pseudoviruses to a lesser extent. Env-specific T cell responses were also induced in mice following Ad4Env160 vector immunization.The Ad4Env vaccine vectors express high levels of Env glycoprotein and induce both Env-specific humoral and cellular immunity thus supporting further development of this new Ad4 HIV-1 Env vaccine platform in Phase 1 clinical trials

Mice immunized with Ad4-H5-Vtn vaccine presented with a reduction of H5N1 reassortant virus in the lungs.

<p>Groups of mice were immunized with Ad4wt virus to establish pre-existing immunity as previously described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031177#s2" target="_blank">Materials and Methods</a> section. Mice were subsequently immunized intranasally with a dose titration of the Ad4-H5-Vtn vaccine. Six weeks following Ad4-H5-Vtn vaccine immunization, the mice were challenged with a lethal dose of H5N1 reassortant virus. Lungs were recovered from a subset of mice 5 days post-challenge to determine influenza-specific viral titers.</p

Ad4-H5-Vtn virus infection of A549 cells induced H5 HA protein expression as detected by Western blot and flow cytometry analysis.

<p>In the case of western blot analysis (A), A549 cells were infected in suspension with clarified crude lysate of Ad4-H5-Vtn virus from the 14^th passage. The cell extracts, 2.5% and 5%, were subjected to SDS-PAGE in 4–12% Bis-Tris gel, blotted onto nitrocellulose membrane and probed with anti-1194 H5HA polyclonal sheep antibody. Lysates as indicated, from uninfected A549 cells were used as negative control. Recombinant His tagged H5HA1203/2004 100 ng and 200 ng proteins were used as positive controls. β-actin was used as loading control. HA₀ refers to full length H5HA; HA₁ and HA₂ represent the proteolytically cleaved form of HA. In the case of flow cytometry (B), A549 cells were infected with a dose titration (vp/mL) of the Ad4-H5-Vtn vector: dotted line, 5×10⁶; solid line, 5×10⁷; dashed line, 5×10⁸. Infected cells were removed and subsequently incubated with primary anti-H5HA and secondary goat anti-IgG PE antibodies. A negative control, A549 cells infected with Ad4wt virus (5×10⁸ vp/mL) was included; grey fill.</p

Ad4-H5-Vtn vector design.

<p>The H5HA native coding sequence, with the polybasic domain removed (B), was derived from A/Vietnam/1194/2004 influenza virus and inserted into the Ad4 virus E3 gene region. The Ad4 virus E3 24.8K, E3 6.3K and E3 29.7K genes were deleted to accommodate the HA transgene and the splice acceptor site of E3 24.8K was retained to drive expression of the HA transgene. The E3A polyadenylation signal sequence, derived from Ad5, was placed downstream of the HA coding sequence. The use of a shuttle plasmid encoding the H5HA sequence and the Ad4 plasmid to obtain the final vaccine product is described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031177#s2" target="_blank">Materials and Methods</a>.</p

Ad4-H5-Vtn virus growth is attenuated in various human cell lines versus Ad4wt virus.

<p>Growth of Ad4-H5-Vtn virus was compared to growth of Ad4wt virus in several human cell lines: A549 a lung carcinoma cell line (A); Hu Tu 80 a duodenum adenocarcinoma cell line (B); MRC-5 an embryonic lung fibroblast cell line (C); H1299 a lung carcinoma line (D); and HepG2 a hepatocellular carcinoma cell line (E). Virus infection and cell growth conditions are described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031177#s2" target="_blank">Materials and Methods</a>.</p

Vaccine-induced H5HA-specific humoral response in the presence and absence of pre-existing Ad4-specific immunity.

<p>Mice were immunized i.n. with 1×10⁹ vp of Ad4wt virus per mouse to establish pre-existing immunity to the vector. Four weeks following the immunization, ten individual mice were bled and Ad4-specific neutralizing antibody titers were determined (A). Mice immunized with Ad4wt virus and naïve mice were subsequently immunized i.n. with a dose titration of the Ad4-H5-Vtn vaccine; 1×10⁹, 1×10⁸, 1×10⁷ and 1×10⁶ vp per mouse and bled 6 weeks after vaccine immunization and again 5 days later following H5N1 reassortant virus challenge to determine HAI antibody titers (B). The immune responses are represented by post-Ad4-H5-Vtn vaccine immunization (open bar) and post-H5N1 reassortant challenge (cumulative of open and black fill bar). Three mice from the group were bled and sera pooled to determine HAI antibody titers.</p

Vaccine-induced H5HA-specific cellular response in the presence and absence of pre-existing Ad4-specific immunity.

<p>Mice were immunized i.n. with 1×10⁹ vp of Ad4wt virus per mouse to establish pre-existing immunity to the vector as previously stated. Two mice were sacrificed and splenocytes pooled to determine Ad4wt virus-specific cellular immunity, as assayed by IFN-γ ELISPOT (A). Two mice were also sacrificed 6 weeks after vaccine immunization and again 5 days later following H5N1 reassortant virus challenge and splenocytes pooled to determine H5 HA-specific cellular immunity evaluated by IFN-γ ELISPOT specific for four H5HA-derived 15-mer peptides, stimulating peptides for ELISPOT response (B). In the case of part (B), the immune responses are represented by post-Ad4-H5-Vtn vaccine immunization (open bar) and post-H5N1 reassortant challenge (cumulative of open and black fill bar). Mice pre-treated with Ad4wt virus and challenged with H5N1 reassortant virus demonstrated no detectable H5HA-specific cellular responses 5 days post-influenza virus challenge. An asterisk * denotes significant IFN-γ responses, <i>p</i><0.05. In the case of two asterisks (*/*) associated with a bar, the bottom and top asterisks refer to post-immunization and post-reassortant H5N1 virus challenge, respectively.</p