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

Oral priming with replicating adenovirus serotype 4 followed by subunit H5N1 vaccine boost promotes antibody affinity maturation and expands H5N1 cross-clade neutralization.

A Phase I trial conducted in 2009-2010 demonstrated that oral vaccination with a replication competent Ad4-H5 (A/Vietnam) vector with dosages ranging from 107-1011 viral particles was well tolerated. HA-specific T-cell responses were efficiently induced, but very limited hemagglutination-inhibiting (HI) humoral responses were measured. However, a single boost of Ad4-H5-Vtn vaccinated individuals with a unadjuvanted licensed H5N1 (A/Vietnam) subunit vaccine resulted in superior HI titers compared with unprimed subjects. In the current study, the impact of Ad4-H5 priming on the quality of the polyclonal humoral immune response was evaluated using a real-time kinetics assay by surface plasmon resonance (SPR). Total binding of serum polyclonal antibodies from the Ad4-H5-Vtn primed groups against both homologous H5N1-A/Vietnam/1194/2004 (clade 1) and heterologous A/Indonesia-5/2005 (clade 2.1) HA1 head domain was significantly higher compared with sera from individuals that received subunit H5N1 vaccination alone. SPR measurements also demonstrated that the antigen-antibody complex dissociation rates (a surrogate for antibody affinity) of serum antibodies against the HA1 of H5N1-A/Vietnam were significantly higher in the Ad4-H5 primed groups compared with those from the unprimed group. Furthermore, strong correlations were observed between the antibody affinities for HA1 (but not HA2) and the virus neutralization titers against the homologous strain and a panel of heterologous clade 2 H5N1 strains. These findings support the concept of oral prime-boost vaccine approaches against pandemic influenza to elicit long-term memory B cells with high affinity capable of rapid response to variant pandemic viruses likely to emerge and adapt to human transmissions

Serum antibody off-rates to H5-Viet-rHA1 following prime-boost strongly correlates with the <i>in-vitro</i> cross-neutralizing capacity against diverse H5N1 strains.

<p>End-point virus neutralization titers of samples collected after the H5N1 subunit vaccine boost are plotted on the X-axis. Antibody off-rate constants that describe the fraction of antibody-antigen complexes decaying per second were determined directly from the serum sample interaction with rHA1 protein using SPR in the dissociation phase are shown on Y-axis. Serum samples from low-dose Ad4-H5-Vtn primed (red circles), high-dose Ad4-H5-Vtn primed (blue circles) or unprimed (green circles) individuals following H5N1 subunit vaccination are shown. Antibody affinity of post-H5N1 vaccinated human sera against HA1 of H5N1-A/Vietnam/1203/2004 strongly correlated with the <i>in-vitro</i> heterologous MN titers against H5N1 clade 2.1- A/Indonesia/5/2005 (A), clade 2.3.4- A/Anhui/1/2005 (B), clade 2.2- A/Turkey/15/2006 (C), and clade 2.2- A/Egypt/NO3072/2010 (D). Spearman correlations are reported for the calculation of correlations between off-rate and MN titers combined across all vaccine groups.</p

Binding of post-H5N1 vaccination polyclonal human serum to properly folded HA1 proteins from A/Vietnam/1203/2004 and A/Indonesia/05/2005.

<p>Steady-state equilibrium analysis of the total binding antibodies in the polyclonal human vaccine sera to properly folded functional H5N1-A/Vietnam/1203/2004 HA1-His₆ (panel A) or H5N1-A/Indonesia/05/2005-His₆ (Panel B) was measured by SPR. Each individual post-boost H5N1 serum sample, diluted ten-fold, was injected simultaneously onto HA1 immobilized on a sensor chip through the free amine group and onto a blank flow cell, free of peptide. Maximum resonance unit (Max RU) values for HA1 binding by serum antibodies obtained from multiple individuals from either low dose (10⁷ VP) Ad4-H5-Vtn primed (red circles), high dose Ad4-H5-Vtn (10¹¹VP) (blue circle), or unprimed (green circles) on day 0 (Pre), 28 days after the third prime (P-P), and 28 days post boost with 90 μg HA/dose of the Sanofi Pasteur (P-V). Differences between groups (p-values) were examined for statistical significance by the multiple comparison adjustment using Bonferroni method. A <i>p-value</i> less than 0.05 was considered to be significant. ‘ns’ represents non-significant (<i>p = >0.05</i>).</p

Serum antibody off-rates to H5-Viet-rHA1 (but not rHA2) following heterologous prime-boost strongly correlate with the <i>in-vitro</i> neutralizing capacity against the homologous H5 vaccine viruses.

<p>Antibody off-rate constants were determined directly from the serum sample interaction with H5N1 rHA1 or rHA2 proteins using SPR in the dissociation phase. SPR analysis of post-boost vaccination human sera from all 3 vaccine groups combined was performed with rHA1 (A) or rHA2 (B) of the H5N1-A/Vietnam/1203/2004 strain, or rHA1 of the heterologous H5N1-A/Indonesia/5/2005 (Clade 2.1) strain (C). Each symbol represents one individual. Serum samples on day 28 following single H5N1 booster vaccination with the subunit H5N1 vaccine (Sanofi Pasteur, 90 μg HA/dose) from the low-dose Ad4-H5-Vtn adjuvanted primed (red circles), high-dose Ad4-H5-Vtn primes (blue circles), or unprimed (green circles) are shown. Antibody affinity of post-H5N1 vaccinated human sera against HA1 (but not HA2) of H5N1-A/Vietnam/1203/2004 correlated with the homologous virus microneutralization titers (MN) against the A/Vietnam/1203/2004 (H5N1). Similarly, polyclonal antibody affinity of post-H5N1 vaccinated human sera against HA1 of the heterologous H5N1-A/Indonesia/5/2005 (Clade 2.1) strain correlated with the microneutralization titers (MN) against the A/Indonesia/5/2005 (H5N1) virus. Spearman correlations are reported for the calculation of correlations between off-rate and MN titers combined across all vaccine groups.</p

H5N1 prime-boost vaccine trial design.

<p>The vaccine study design representing the three groups that were used in the current study and the individual numbers (N) per group is indicated. For more details of the clinical study see Reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115476#pone.0115476.ref003" target="_blank">3</a>. Ad4-H5N1-Viet and MIV-H5N1-Viet refer to: Ad4-H5-Vtn is a recombinant, replication competent Ad4, encoding full-length haemagglutinin from influenza A H5N1 virus (A/Vietnam/1194/2004) given orally thrice either as 10⁷ virus particles (VP) or 10¹¹ virus particles per dose. MIV-H5N1-Viet is a formalin-inactivated, licensed inactivated H5N1 partially purified subunit vaccine (A/Vietnam, 90 μg HA/dose; Sanofi Pasteur).</p