T cell responses induced by adenoviral vectored vaccines can be adjuvanted by fusion of antigen to the oligomerization domain of C4b-binding protein.

Viral vectored vaccines have been shown to induce both T cell and antibody responses in animals and humans. However, the induction of even higher level T cell responses may be crucial in achieving vaccine efficacy against difficult disease targets, especially in humans. Here we investigate the oligomerization domain of the α-chain of C4b-binding protein (C4 bp) as a candidate T cell "molecular adjuvant" when fused to malaria antigens expressed by human adenovirus serotype 5 (AdHu5) vectored vaccines in BALB/c mice. We demonstrate that i) C-terminal fusion of an oligomerization domain can enhance the quantity of antigen-specific CD4(+) and CD8(+) T cell responses induced in mice after only a single immunization of recombinant AdHu5, and that the T cells maintain similar functional cytokine profiles; ii) an adjuvant effect is observed for AdHu5 vectors expressing either the 42 kDa C-terminal domain of Plasmodium yoelii merozoite surface protein 1 (PyMSP1(42)) or the 83 kDa ectodomain of P. falciparum strain 3D7 apical membrane antigen 1 (PfAMA1), but not a candidate 128kDa P. falciparum MSP1 biallelic fusion antigen; iii) following two homologous immunizations of AdHu5 vaccines, antigen-specific T cell responses are further enhanced, however, in both BALB/c mice and New Zealand White rabbits no enhancement of functional antibody responses is observed; and iv) that the T cell adjuvant activity of C4 bp is not dependent on a functional Fc-receptor γ-chain in the host, but is associated with the oligomerization of small (<80 kDa) antigens expressed by recombinant AdHu5. The oligomerization domain of C4 bp can thus adjuvant T cell responses induced by AdHu5 vectors against selected antigens and its clinical utility as well as mechanism of action warrant further investigation

Preclinical assessment of viral vectored and protein vaccines targeting the Duffy-binding protein region II of Plasmodium vivax

Malaria vaccine development has largely focused on Plasmodium falciparum; however, a reawakening to the importance of Plasmodium vivax has spurred efforts to develop vaccines against this difficult to treat and at times severe form of relapsing malaria, which constitutes a significant proportion of human malaria cases worldwide. The almost complete dependence of P. vivax red blood cell invasion on the interaction of the P. vivax Duffy-binding protein region II (PvDBP_RII) with the human Duffy antigen receptor for chemokines (DARC) makes this antigen an attractive vaccine candidate against blood-stage P. vivax. Here, we generated both preclinical and clinically compatible adenoviral and poxviral vectored vaccine candidates expressing the Salvador I allele of PvDBP_RII – including human adenovirus serotype 5 (HAdV5), chimpanzee adenovirus serotype 63 (ChAd63), and modified vaccinia virus Ankara (MVA) vectors. We report on the antibody and T cell immunogenicity of these vaccines in mice or rabbits, either used alone in a viral vectored prime-boost regime or in “mixed-modality” adenovirus prime – protein-in-­adjuvant boost regimes (using a recombinant PvDBP_RII protein antigen formulated in Montanide®ISA720 or Abisco®100 adjuvants). Antibodies induced by these regimes were found to bind to native parasite antigen from P. vivax infected Thai patients and were capable of inhibiting the binding of PvDBP_RII to its receptor DARC using an in vitro binding inhibition assay. In recent years, recombinant ChAd63 and MVA vectors have been quickly translated into human clinical trials for numerous antigens from P. falciparum as well as a growing number of other pathogens. The vectors reported here are immunogenic in small animals, elicit antibodies against PvDBP_RII, and have recently entered clinical trials, which will provide the first assessment of the safety and immunogenicity of the PvDBP_RII antigen in humans

Assessment of humoral immune responses to blood-stage malaria antigens following ChAd63-MVA immunization, controlled human malaria infection and natural exposure.

The development of protective vaccines against many difficult infectious pathogens will necessitate the induction of effective antibody responses. Here we assess humoral immune responses against two antigens from the blood-stage merozoite of the Plasmodium falciparum human malaria parasite--MSP1 and AMA1. These antigens were delivered to healthy malaria-naïve adult volunteers in Phase Ia clinical trials using recombinant replication-deficient viral vectors--ChAd63 to prime the immune response and MVA to boost. In subsequent Phase IIa clinical trials, immunized volunteers underwent controlled human malaria infection (CHMI) with P. falciparum to assess vaccine efficacy, whereby all but one volunteer developed low-density blood-stage parasitemia. Here we assess serum antibody responses against both the MSP1 and AMA1 antigens following i) ChAd63-MVA immunization, ii) immunization and CHMI, and iii) primary malaria exposure in the context of CHMI in unimmunized control volunteers. Responses were also assessed in a cohort of naturally-immune Kenyan adults to provide comparison with those induced by a lifetime of natural malaria exposure. Serum antibody responses against MSP1 and AMA1 were characterized in terms of i) total IgG responses before and after CHMI, ii) responses to allelic variants of MSP1 and AMA1, iii) functional growth inhibitory activity (GIA), iv) IgG avidity, and v) isotype responses (IgG1-4, IgA and IgM). These data provide the first in-depth assessment of the quality of adenovirus-MVA vaccine-induced antibody responses in humans, along with assessment of how these responses are modulated by subsequent low-density parasite exposure. Notable differences were observed in qualitative aspects of the human antibody responses against these malaria antigens depending on the means of their induction and/or exposure of the host to the malaria parasite. Given the continued clinical development of viral vectored vaccines for malaria and a range of other diseases targets, these data should help to guide further immuno-monitoring studies of vaccine-induced human antibody responses

Functional comparison of blood-stage Plasmodium falciparum malaria vaccine candidate antigens

The malaria genome encodes over 5,000 proteins and many of these have also been proposed to be potential vaccine candidates, although few of these have been tested clinically. RH5 is one of the leading blood-stage Plasmodium falciparum malaria vaccine antigens and Phase I/II clinical trials of vaccines containing this antigen are currently underway. Its likely mechanism of action is to elicit antibodies that can neutralize merozoites by blocking their invasion of red blood cells (RBC). However, many other antigens could also elicit neutralizing antibodies against the merozoite, and most of these have never been compared directly to RH5. The objective of this study was to compare a range of blood-stage antigens to RH5, to identify any antigens that outperform or synergize with anti-RH5 antibodies. We selected 55 gene products, covering 15 candidate antigens that have been described in the literature and 40 genes selected on the basis of bioinformatics functional prediction. We were able to make 20 protein-in-adjuvant vaccines from the original selection. Of these, S-antigen and CyRPA robustly elicited antibodies with neutralizing properties. Anti-CyRPA IgG generally showed additive GIA with anti-RH5 IgG, although high levels of anti-CyRPA-specific rabbit polyclonal IgG were required to achieve 50% GIA. Our data suggest that further vaccine antigen screening efforts are required to identify a second merozoite target with similar antibody-susceptibility to RH5

Analysis of human B‐cell responses following ChAd63‐MVA MSP1 and AMA1 immunization and controlled malaria infection

Acquisition of non‐sterilizing natural immunity to Plasmodium falciparum malaria has been shown in low transmission areas following multiple exposures. However, conflicting data from endemic areas suggest that the parasite may interfere with the induction of effective B‐cell responses. To date, the impact of blood‐stage parasite exposure on antigen‐specific B cells has not been reported following controlled human malaria infection (CHMI). Here we analysed human B‐cell responses in a series of Phase I/IIa clinical trials, which include CHMI, using candidate virus‐vectored vaccines encoding two blood‐stage antigens: merozoite surface protein 1 (MSP1) and apical membrane antigen 1 (AMA1). Previously vaccinated volunteers show boosting of pre‐existing antigen‐specific memory B‐cell (mBC) responses following CHMI. In contrast, unvaccinated malaria‐naive control volunteers developed an mBC response against MSP1 but not AMA1. Serum IgG correlated with the mBC response after booster vaccination but this relationship was less well maintained following CHMI. A significant reduction in peripheral MSP1‐specific mBC was observed at the point of diagnosis of blood‐stage infection. This was coincident with a reduction in peripheral blood B‐cell subsets expressing CXCR3 and elevated serum levels of interferon‐γ and CXCL9, suggesting migration away from the periphery. These CHMI data confirm that mBC and antibody responses can be induced and boosted by blood‐stage parasite exposure, in support of epidemiological studies on low‐level parasite exposure

Germinal Center B Cell and T Follicular Helper Cell Responses to Viral Vector and Protein-in-Adjuvant Vaccines

There is great interest in the development of antibody-inducing subunit vaccines targeting infections including HIV, malaria and Ebola. We previously reported that adenovirus vectored vaccines are potent in priming antibody responses, but uncertainty remains regarding the optimal approach for induction of humoral immune responses. Here, using ovalbumin as a model antigen, we assessed the magnitude of the primary and anamnestic antigen-specific IgG responses of mice to four clinically-relevant vaccine formulations: replication-deficient adenovirus; modified vaccinia Ankara (MVA, a poxvirus); protein with alum; and protein in the squalene oil-in-water adjuvant Addavax. We then used flow cytometric assays capable of measuring total and antigen-specific germinal center (GC) B cell and follicular helper T cell (Tfh) responses to compare the induction of these responses by the different formulations. We report that adenovirus vectored vaccines induce antigen insert-specific GC B cell and antibody responses of a magnitude comparable to those induced by a potent protein/ squalene oil-in-water formulation whereas- despite a robust overall GC response - the insertspecific GC B cell and antibody responses induced by MVA were extremely weak. Antigenspecific Tfh responses to adenovirus vectored responses exceeded those induced by other platforms at day 7 after immunization. We found little evidence that innate immune activation by adenovirus may act as an adjuvant in such a manner that the humoral response to a recombinant protein may be enhanced by co-administering with an adenovirus lacking a transgene of interest. Overall, these studies provide further support for the use of replication-deficient adenoviruses to induce humoral responses

Germinal centre B cell and T follicular helper cell responses to viral vector and protein-in-adjuvant vaccines

There is great interest in the development of antibody-inducing subunit vaccines targeting infections including HIV, malaria and Ebola. We previously reported that adenovirus vectored vaccines are potent in priming antibody responses, but uncertainty remains regarding the optimal approach for induction of humoral immune responses. Here, using ovalbumin as a model antigen, we assessed the magnitude of the primary and anamnestic antigen-specific IgG responses of mice to four clinically-relevant vaccine formulations: replication-deficient adenovirus; modified vaccinia Ankara (MVA, a poxvirus); protein with alum; and protein in the squalene oil-in-water adjuvant Addavax. We then used flow cytometric assays capable of measuring total and antigen-specific germinal center (GC) B cell and follicular helper T cell (Tfh) responses to compare the induction of these responses by the different formulations. We report that adenovirus vectored vaccines induce antigen insert-specific GC B cell and antibody responses of a magnitude comparable to those induced by a potent protein/ squalene oil-in-water formulation whereas- despite a robust overall GC response - the insertspecific GC B cell and antibody responses induced by MVA were extremely weak. Antigenspecific Tfh responses to adenovirus vectored responses exceeded those induced by other platforms at day 7 after immunization. We found little evidence that innate immune activation by adenovirus may act as an adjuvant in such a manner that the humoral response to a recombinant protein may be enhanced by co-administering with an adenovirus lacking a transgene of interest. Overall, these studies provide further support for the use of replication-deficient adenoviruses to induce humoral responses

Investigating the Induction of Vaccine-Induced Th17 and Regulatory T Cells in Healthy, Mycobacterium bovis BCG Immunized Adults Vaccinated with a New Tuberculosis Vaccine, MVA85A

Tuberculosis (TB) remains a threat to global health. While advances in diagnostics and treatment are crucial to the containment of the epidemic, it is likely that elimination of the disease can only be achieved through vaccination. Vaccine-induced protection from Mycobacterium tuberculosis is dependent, at least in part, on a robust Th1 response, yet little is known of the ability of TB vaccines to induce other T-cell subsets which may influence vaccine efficacy. Interleukin-17A (IL-17A) is a proinflammatory cytokine produced by Th17 cells which has been associated with both immune pathology and protection against infectious disease. Following vaccination with MVA85A, a viral vector vaccine aimed at enhancing immune responses to M. tuberculosis, antigen-specific IL-17A-producing T cells were induced in the peripheral blood of healthy volunteers. These T cells are detected later than gamma interferon (IFN-gamma)-secreting T cells and are of a low magnitude. Preexisting immune responses to mycobacterial antigens were associated with higher CD4(+) CD25(hi) CD39(+) T-cell levels in the periphery and a reduced capacity to produce IL-17A following immunization. These data highlight the intricate balance of effector and regulatory immune responses induced by vaccination and that preexisting immunity to mycobacterial antigens may affect the composition of vaccine-induced T-cell subsets

The utility of Plasmodium berghei as a rodent model for anti-merozoite malaria vaccine assessment.

Rodent malaria species Plasmodium yoelii and P. chabaudi have been widely used to validate vaccine approaches targeting blood-stage merozoite antigens. However, increasing data suggest the P. berghei rodent malaria may be able to circumvent vaccine-induced anti-merozoite responses. Here we confirm a failure to protect against P. berghei, despite successful antibody induction against leading merozoite antigens using protein-in-adjuvant or viral vectored vaccine delivery. No subunit vaccine approach showed efficacy in mice following immunization and challenge with the wild-type P. berghei strains ANKA or NK65, or against a chimeric parasite line encoding a merozoite antigen from P. falciparum. Protection was not improved in knockout mice lacking the inhibitory Fc receptor CD32b, nor against a Δsmac P. berghei parasite line with a non-sequestering phenotype. An improved understanding of the mechanisms responsible for protection, or failure of protection, against P. berghei merozoites could guide the development of an efficacious vaccine against P. falciparum

Preclinical assessment of viral vectored and protein vaccines targeting the Duffy-binding protein region II of Plasmodium vivax

Malaria vaccine development has largely focused on Plasmodium falciparum; however a reawakening to the importance of P. vivax has spurred efforts to develop vaccines against this difficult to treat and at times severe form of relapsing malaria, which constitutes a significant proportion of human malaria cases worldwide. The almost complete dependence of P. vivax red blood cell invasion on the interaction of the P. vivax Duffy-binding protein region II (PvDBP_RII) with the human Duffy antigen receptor for chemokines (DARC), makes this antigen an attractive vaccine candidate against blood-stage P. vivax. Here, we generated both preclinical and clinically-compatible adenoviral and poxviral vectored vaccine candidates expressing the Salvador I allele of PvDBP_RII – including human adenovirus serotype 5 (HAdV5), chimpanzee adenovirus serotype 63 (ChAd63) and modified vaccinia virus Ankara (MVA) vectors. We report on the antibody and T cell immunogenicity of these vaccines in mice or rabbits, either used alone in a viral vectored prime-boost regime, or in ‘mixed-modality’ adenovirus prime – protein-in-adjuvant boost regimes (using a recombinant protein PvDBP_RII protein antigen formulated in Montanide®ISA720 or Abisco®100 adjuvants). Antibodies induced by these regimes were found to bind to native parasite antigen from P. vivax infected Thai patients and were capable of inhibiting the binding of PvDBP_RII to its receptor DARC using an in vitro binding inhibition assay. In recent years, recombinant ChAd63 and MVA vectors have been quickly translated into human clinical trials for numerous antigens from P. falciparum as well as a growing number of other pathogens. The vectors reported here are immunogenic in small animals, elicit antibodies against PvDBP_RII and have recently entered clinical trials which will provide the first assessment of the safety and immunogenicity of the PvDBP_RII antigen in humans