Matthew D. J. Dicks

Phase Ia clinical evaluation of the Plasmodium falciparum blood-stage antigen MSP1 in ChAd63 and MVA vaccine vectors.

Efficacy trials of antibody-inducing protein-in-adjuvant vaccines targeting the blood-stage Plasmodium falciparum malaria parasite have so far shown disappointing results. The induction of cell-mediated responses in conjunction with antibody responses is thought to be one alternative strategy that could achieve protective efficacy in humans. Here, we prepared chimpanzee adenovirus 63 (ChAd63) and modified vaccinia virus Ankara (MVA) replication-deficient vectors encoding the well-studied P. falciparum blood-stage malaria antigen merozoite surface protein 1 (MSP1). A phase Ia clinical trial was conducted in healthy adults of a ChAd63-MVA MSP1 heterologous prime-boost immunization regime. The vaccine was safe and generally well tolerated. Fewer systemic adverse events (AEs) were observed following ChAd63 MSP1 than MVA MSP1 administration. Exceptionally strong T-cell responses were induced, and these displayed a mixed of CD4(+) and CD8(+) phenotype. Substantial MSP1-specific serum immunoglobulin G (IgG) antibody responses were also induced, which were capable of recognizing native parasite antigen, but these did not reach titers sufficient to neutralize P. falciparum parasites in vitro. This viral vectored vaccine regime is thus a leading approach for the induction of strong cellular and humoral immunogenicity against difficult disease targets in humans. Further studies are required to assess whether this strategy can achieve protective efficacy against blood-stage malaria infection.

Phase Ia clinical evaluation of the safety and immunogenicity of the Plasmodium falciparum blood-stage antigen AMA1 in ChAd63 and MVA vaccine vectors.

Background Traditionally, vaccine development against the blood-stage of Plasmodium falciparum infection has focused on recombinant protein-adjuvant formulations in order to induce high-titer growth-inhibitory antibody responses. However, to date no such vaccine encoding a blood-stage antigen(s) alone has induced significant protective efficacy against erythrocytic-stage infection in a pre-specified primary endpoint of a Phase IIa/b clinical trial designed to assess vaccine efficacy. Cell-mediated responses, acting in conjunction with functional antibodies, may be necessary for immunity against blood-stage P. falciparum. The development of a vaccine that could induce both cell-mediated and humoral immune responses would enable important proof-of-concept efficacy studies to be undertaken to address this question. Methodology We conducted a Phase Ia, non-randomized clinical trial in 16 healthy, malaria-naïve adults of the chimpanzee adenovirus 63 (ChAd63) and modified vaccinia virus Ankara (MVA) replication-deficient viral vectored vaccines encoding two alleles (3D7 and FVO) of the P. falciparum blood-stage malaria antigen; apical membrane antigen 1 (AMA1). ChAd63-MVA AMA1 administered in a heterologous prime-boost regime was shown to be safe and immunogenic, inducing high-level T cell responses to both alleles 3D7 (median 2036 SFU/million PBMC) and FVO (median 1539 SFU/million PBMC), with a mixed CD4+/CD8+ phenotype, as well as substantial AMA1-specific serum IgG responses (medians of 49 µg/mL and 41 µg/mL for 3D7 and FVO AMA1 respectively) that demonstrated growth inhibitory activity in vitro. Conclusions ChAd63-MVA is a safe and highly immunogenic delivery platform for both alleles of the AMA1 antigen in humans which warrants further efficacy testing. ChAd63-MVA is a promising heterologous prime-boost vaccine strategy that could be applied to numerous other diseases where strong cellular and humoral immune responses are required for protection. Trial Registration ClinicalTrials.gov NCT01095055

Enhancing Blood-Stage Malaria Subunit Vaccine Immunogenicity in Rhesus Macaques by Combining Adenovirus, Poxvirus, and Protein-in-Adjuvant Vaccines

Protein-in-adjuvant formulations and viral-vectored vaccines encoding blood-stage malaria Ags have shown efficacy in rodent malaria models and in vitro assays against Plasmodium falciparum. Abs and CD4+ T cell responses are associated with protective efficacy against blood-stage malaria, whereas CD8+ T cells against some classical blood-stage Ags can also have a protective effect against liver-stage parasites. No subunit vaccine strategy alone has generated demonstrable high-level efficacy against blood-stage infection in clinical trials. The induction of high-level Ab responses, as well as potent T and B cell effector and memory populations, is likely to be essential to achieve immediate and sustained protective efficacy in humans. This study describes in detail the immunogenicity of vaccines against P. falciparum apical membrane Ag 1 in rhesus macaques (Macaca mulatta), including the chimpanzee adenovirus 63 (AdCh63), the poxvirus modified vaccinia virus Ankara (MVA), and protein vaccines formulated in Alhydrogel or CoVaccine HT adjuvants. AdCh63-MVA heterologous prime-boost immunization induces strong and long-lasting multifunctional CD8+ and CD4+ T cell responses that exhibit a central memory-like phenotype. Three-shot (AdCh63-MVA-protein) or two-shot (AdCh63-protein) regimens induce memory B cells and high-titer functional IgG responses that inhibit the growth of two divergent strains of P. falciparum in vitro. Prior immunization with adenoviral vectors of alternative human or simian serotype does not affect the immunogenicity of the AdCh63 apical membrane Ag 1 vaccine. These data encourage the further clinical development and coadministration of protein and viral vector vaccine platforms in an attempt to induce broad cellular and humoral immune responses against blood-stage malaria Ags in humans.

A Novel Chimpanzee Adenovirus Vector with Low Human Seroprevalence: Improved Systems for Vector Derivation and Comparative Immunogenicity

Recombinant adenoviruses are among the most promising tools for vaccine antigen delivery. Recently, the development of new vectors has focused on serotypes to which the human population is less exposed in order to circumvent pre-existing anti vector immunity. This study describes the derivation of a new vaccine vector based on a chimpanzee adenovirus, Y25, together with a comparative assessment of its potential to elicit transgene product specific immune responses in mice. The vector was constructed in a bacterial artificial chromosome to facilitate genetic manipulation of genomic clones. In order to conduct a fair head-to-head immunological comparison of multiple adenoviral vectors, we optimised a method for accurate determination of infectious titre, since this parameter exhibits profound natural variability and can confound immunogenicity studies when doses are based on viral particle estimation. Cellular immunogenicity of recombinant E1 E3-deleted vector ChAdY25 was comparable to that of other species E derived chimpanzee adenovirus vectors including ChAd63, the first simian adenovirus vector to enter clinical trials in humans. Furthermore, the prevalence of virus neutralizing antibodies (titre >1∶200) against ChAdY25 in serum samples collected from two human populations in the UK and Gambia was particularly low compared to published data for other chimpanzee adenoviruses. These findings support the continued development of new chimpanzee adenovirus vectors, including ChAdY25, for clinical use.

Clinical Assessment of a Novel Recombinant Simian Adenovirus ChAdOx1 as a Vectored Vaccine Expressing Conserved Influenza A Antigens

Adenoviruses are potent vectors for inducing and boosting cellular immunity to encoded recombinant antigens. However, the widespread seroprevalence of neutralizing antibodies to common human adenovirus serotypes limits their use. Simian adenoviruses do not suffer from the same drawbacks. We have constructed a replication-deficient chimpanzee adenovirus-vectored vaccine expressing the conserved influenza antigens, nucleoprotein (NP), and matrix protein 1 (M1). Here, we report safety and T-cell immunogenicity following vaccination with this novel recombinant simian adenovirus, ChAdOx1 NP+M1, in a first in human dose-escalation study using a 3+3 study design, followed by boosting with modified vaccinia virus Ankara expressing the same antigens in some volunteers. We demonstrate ChAdOx1 NP+M1 to be safe and immunogenic. ChAdOx1 is a promising vaccine vector that could be used to deliver vaccine antigens where strong cellular immune responses are required for protection.

Tailoring subunit vaccine immunogenicity: Maximizing antibody and T cell responses by using combinations of adenovirus, poxvirus and protein-adjuvant vaccines against Plasmodium falciparum MSP1

Subunit vaccination modalities tend to induce particular immune effector responses. Viral vectors are well known for their ability to induce strong T cell responses, while protein-adjuvant vaccines have been used primarily for induction of antibody responses. Here, we demonstrate in mice using a Plasmodium falciparum merozoite surface protein 1 (PfMSP1) antigen that novel regimes combining adenovirus and poxvirus vectored vaccines with protein antigen in Montanide ISA720 adjuvant can achieve simultaneous antibody and T cell responses which equal, or in some cases surpass, the best immune responses achieved by either the viral vectors or the protein vaccine alone. Such broad responses can be achieved either using three-stage vaccination protocols, or with an equally effective two-stage protocol in which viral vectors are admixed with protein and adjuvant, and were apparent despite the use of a protein antigen that represented only a portion of the viral vector antigen. We describe further possible advantages of viral vectors in achieving consistent antibody priming, enhanced antibody avidity, and cytophilic isotype skew. These data strengthen the evidence that tailored combinations of vaccine platforms can achieve desired combinations of immune responses, and further encourage the co-administration of antibody-inducing recombinant protein vaccines with T cell- and antibody-inducing recombinant viral vectors as one strategy that may achieve protective blood-stage malaria immunity in humans.

New Candidate Vaccines against Blood-Stage Plasmodium falciparum Malaria: Prime-Boost Immunization Regimens Incorporating Human and Simian Adenoviral Vectors and Poxviral Vectors Expressing an Optimized Antigen Based on Merozoite Surface Protein 1

ABSTRACT Although merozoite surface protein 1 (MSP-1) is a leading candidate vaccine antigen for blood-stage malaria, its efficacy in clinical trials has been limited in part by antigenic polymorphism and potentially by the inability of protein-in-adjuvant vaccines to induce strong cellular immunity. Here we report the design of novel vectored Plasmodium falciparum vaccines capable of overcoming such limitations. We optimized an antigenic insert comprising the four conserved blocks of MSP-1 fused to tandemly arranged sequences that represent both allelic forms of the dimorphic 42-kDa C-terminal region. Inserts were expressed by adenoviral and poxviral vectors and employed in heterologous prime-boost regimens. Simian adenoviral vectors were used in an effort to circumvent preexisting immunity to human adenoviruses. In preclinical studies these vaccines induced potent cellular immune responses and high-titer antibodies directed against MSP-1. The antibodies induced were found to have growth-inhibitory activity against dimorphic allelic families of P. falciparum. These vectored vaccines should allow assessment in humans of the safety and efficacy of inducing strong cellular as well as cross-strain humoral immunity to P. falciparum MSP-1.

Transgene Optimization, Immunogenicity and In Vitro Efficacy of Viral Vectored Vaccines Expressing Two Alleles of Plasmodium falciparum AMA1

Background Apical membrane antigen 1 (AMA1) is a leading candidate vaccine antigen against blood-stage malaria, although to date numerous clinical trials using mainly protein-in-adjuvant vaccines have shown limited success. Here we describe the pre-clinical development and optimization of recombinant human and simian adenoviral (AdHu5 and ChAd63) and orthopoxviral (MVA) vectors encoding transgene inserts for Plasmodium falciparum AMA1 (PfAMA1). Methodology/Principal Findings AdHu5-MVA prime-boost vaccination in mice and rabbits using these vectors encoding the 3D7 allele of PfAMA1 induced cellular immune responses as well as high-titer antibodies that showed growth inhibitory activity (GIA) against the homologous but not heterologous parasite strains. In an effort to overcome the issues of PfAMA1 antigenic polymorphism and pre-existing immunity to AdHu5, a simian adenoviral (ChAd63) vector and MVA encoding two alleles of PfAMA1 were developed. This antigen, composed of the 3D7 and FVO alleles of PfAMA1 fused in tandem and with expression driven by a single promoter, was optimized for antigen secretion and transmembrane expression. These bi-allelic PfAMA1 vaccines, when administered to mice and rabbits, demonstrated comparable immunogenicity to the mono-allelic vaccines and purified serum IgG now showed GIA against the two divergent strains of P. falciparum encoded in the vaccine. CD8+ and CD4+ T cell responses against epitopes that were both common and unique to the two alleles of PfAMA1 were also measured in mice. Conclusions/Significance Optimized transgene inserts encoding two divergent alleles of the same antigen can be successfully inserted into adeno- and pox-viral vaccine vectors. Adenovirus-MVA immunization leads to the induction of T cell responses common to both alleles, as well as functional antibody responses that are effective against both of the encoded strains of P. falciparum in vitro. These data support the further clinical development of these vaccine candidates in Phase I/IIa clinical trials.

Enhancing immunogenicity and transmission-blocking activity of malaria vaccines by fusing Pfs25 to IMX313 multimerization technology.

Transmission-blocking vaccines (TBV) target the sexual-stages of the malaria parasite in the mosquito midgut and are widely considered to be an essential tool for malaria elimination. High-titer functional antibodies are required against target antigens to achieve effective transmission-blocking activity. We have fused Pfs25, the leading malaria TBV candidate antigen to IMX313, a molecular adjuvant and expressed it both in ChAd63 and MVA viral vectors and as a secreted protein-nanoparticle. Pfs25-IMX313 expressed from viral vectors or as a protein-nanoparticle is significantly more immunogenic and gives significantly better transmission-reducing activity than monomeric Pfs25. In addition, we demonstrate that the Pfs25-IMX313 protein-nanoparticle leads to a qualitatively improved antibody response in comparison to soluble Pfs25, as well as to significantly higher germinal centre (GC) responses. These results demonstrate that antigen multimerization using IMX313 is a very promising strategy to enhance antibody responses against Pfs25, and that Pfs25-IMX313 is a highly promising TBV candidate vaccine.

Mixed Vector Immunization With Recombinant Adenovirus and MVA Can Improve Vaccine Efficacy While Decreasing Antivector Immunity

Substantial protection can be provided against the pre-erythrocytic stages of malaria by vaccination first with an adenoviral and then with an modified vaccinia virus Ankara (MVA) poxviral vector encoding the same ME.TRAP transgene. We investigated whether the two vaccine components adenovirus (Ad) and MVA could be coinjected as a mixture to enhance protection against malaria. A single-shot mixture at specific ratios of Ad and MVA (Ad+MVA) enhanced CD8+ T cell-dependant protection of mice against challenge with Plasmodium berghei. Moreover, the degree of protection could be enhanced after homologous boosting with the same Ad+MVA mixture to levels comparable with classic heterologous Ad prime-MVA boost regimes. The mixture increased transgene-specific responses while decreasing the CD8+ T cell antivector immunity compared to each vector used alone, particularly against the MVA backbone. Mixed vector immunization led to increased early circulating interferon-γ (IFN-γ) response levels and altered transcriptional microarray profiles. Furthermore, we found that sequential immunizations with the Ad+MVA mixture led to consistent boosting of the transgene-specific CD8+ response for up to three mixture immunizations, whereas each vector used alone elicited progressively lower responses. Our findings offer the possibility of simplifying the deployment of viral vectors as a single mixture product rather than in heterologous prime-boost regimens.