Development of adjuvanted multi-antigen liver-stage malaria vaccines

Despite promising progress in malaria vaccine development in recent years, an efficacious subunit vaccine against P. falciparum remains to be licensed and deployed. The most advanced candidates in clinical development focus on two pre-erythrocytic antigens (CSP and ME-TRAP), but many more immunogenic antigens have been recently identified. The work described in this thesis aimed to improve on the immunogenicity and efficacy of the leading liver-stage vaccine candidate (ChAd63-MVA ME-TRAP), which is known to confer protection by eliciting high levels of antigen-specific CD8+ T cells. To achieve this, two different strategies were pursued. First, several prime-boost regimens with vectors encoding combinations of two liver-stage antigens were investigated. When mixed, vectors expressing LSA1 and LSAP2 conferred highest levels of protective efficacy in mice and were therefore considered for inclusion in the final second generation viral vectored liver-stage malaria vaccine. Second, the MHC class II invariant chain (Ii) was developed as a molecular adjuvant. Immunogenicity analyses of ChAd63 encoding ME-TRAP fused to several different versions of Ii showed that the transmembrane domain of Ii has the ability to strongly increase antigen-specific CD8+ T-cell responses, even in the absence of the rest of the Ii protein. This finding may lead to the discovery of numerous similar adjuvants (such as the transmembrane domain of the Newcastle disease virus fusion protein). Furthermore, experiments showed that the Ii chain sequence can also be xenogenised without losing adjuvanticity. Both strategies have been combined in numerous novel adjuvanted multi-antigen vaccines, which were ranked for immunogenicity and efficacy in inbred and outbred mice. The viral vectors of the best combination (ChAdOX1-sharkTM/Ii-LSA1-LSAP2 and MVA-tPA-LSA1-LSAP2) are currently being produced to GMP grade and will progress to initial clinical trials in early 2017.</p

Development of adjuvanted multi-antigen liver-stage malaria vaccines

Despite promising progress in malaria vaccine development in recent years, an efficacious subunit vaccine against P. falciparum remains to be licensed and deployed. The most advanced candidates in clinical development focus on two pre-erythrocytic antigens (CSP and ME-TRAP), but many more immunogenic antigens have been recently identified. The work described in this thesis aimed to improve on the immunogenicity and efficacy of the leading liver-stage vaccine candidate (ChAd63-MVA ME-TRAP), which is known to confer protection by eliciting high levels of antigen-specific CD8+ T cells. To achieve this, two different strategies were pursued. First, several prime-boost regimens with vectors encoding combinations of two liver-stage antigens were investigated. When mixed, vectors expressing LSA1 and LSAP2 conferred highest levels of protective efficacy in mice and were therefore considered for inclusion in the final second generation viral vectored liver-stage malaria vaccine. Second, the MHC class II invariant chain (Ii) was developed as a molecular adjuvant. Immunogenicity analyses of ChAd63 encoding ME-TRAP fused to several different versions of Ii showed that the transmembrane domain of Ii has the ability to strongly increase antigen-specific CD8+ T-cell responses, even in the absence of the rest of the Ii protein. This finding may lead to the discovery of numerous similar adjuvants (such as the transmembrane domain of the Newcastle disease virus fusion protein). Furthermore, experiments showed that the Ii chain sequence can also be xenogenised without losing adjuvanticity. Both strategies have been combined in numerous novel adjuvanted multi-antigen vaccines, which were ranked for immunogenicity and efficacy in inbred and outbred mice. The viral vectors of the best combination (ChAdOX1-sharkTM/Ii-LSA1-LSAP2 and MVA-tPA-LSA1-LSAP2) are currently being produced to GMP grade and will progress to initial clinical trials in early 2017.</p

Immunodominant responses to PfUIS3.

<p>(A) BALB/c, (B) C57BL/6 or (C) HLA-A2 tg mice (n = 4 per strain) were vaccinated i.m. with 1x10⁸ infectious units (ifu) ChAd63-PfUIS3 followed eight weeks later by 1x10⁶ plaque forming units (pfu) MVA-PfUIS3. Two weeks post-MVA boost, mice were sacrificed and splenocytes isolated to perform an <i>ex vivo</i> IFNγ ELISpot. Splenocytes were stimulated with either an overlapping peptide pool to PfUIS3 or individual peptides (20aa each, overlapping by ten). Both median and individual data points are shown. For (A) BALB/c and (B) C57BL/6, CD4⁺ and CD8⁺ epitopes were also determined (right panel). Two weeks post-ChAd63 (n = 4 per strain), splenocytes were isolated and incubated with the appropriate peptide for six hours prior to ICS staining. Box plots show the percentage IFNγ⁺ of CD4⁺ or CD8⁺ cells, with whiskers representing the maximum and minimum.</p

Polyfunctionality of CD8⁺ T cells induced by ChAd63-MVA vaccination in BALB/c mice.

<p>BALB/c mice (n = 4) were vaccinated with ChAd63-MVA (A) PfUIS3, (B) PfLSA1 or (C) PfLSAP2, as previously described. Two weeks post-ChAd63 prime and one-week post-MVA boost blood was taken and cellular immunogenicity assessed by ICS, after stimulation for six hours with an overlapping peptide pool to the appropriate antigen. Two weeks post-MVA boost mice were sacrificed, spleens harvested and cellular immunogenicity again assessed by ICS. The proportion of cells at each time-point expressing one, two or three cytokines is shown. The bar chart indicates which cytokines were produced, whilst the slices of the pie chart indicate the proportion of cells producing one (purple), two (orange) or three (black) cytokines.</p

Assessment of the Plasmodium falciparum pre-erythrocytic antigen UIS3 as a potential candidate for a malaria vaccine

Efforts are currently underway to improve the efficacy of sub-unit malaria vaccines through assessment of new adjuvants, vaccination platforms and antigens. In this study, we further assess the antigen P. falciparum (Pf) upregulated in infective sporozoites 3 (PfUIS3) as a vaccine candidate. PfUIS3 was expressed in the viral vectors chimpanzee adenovirus 63 (ChAd63) and modified vaccinia virus Ankara (MVA) and used to immunize mice in a prime-boost regimen. We previously demonstrated that this regimen could provide partial protection against challenge with chimeric P. berghei (Pb) parasites expressing PfUIS3. We now show that ChAd63-MVA PfUIS3 can also provide partial cross-species protection against challenge with wild type Pb parasites. We also show that PfUIS3-specific cellular memory responses can be recalled in human volunteers exposed to Pf parasites in a controlled human malaria infection study. When ChAd63-MVA PfUIS3 was co-administered with the vaccine candidate ChAd63-MVA Pf thrombospondin-related adhesion protein (TRAP), there was no significant change in immunogenicity to either vaccine. However, when these mice were challenged with double chimeric Pb-Pf parasites expressing both PfUIS3 and PfTRAP, vaccine efficacy was improved to 100% sterile protection. This synergistic effect was only evident when the two vaccines were mixed and administered at the same site. We have therefore demonstrated that vaccination with PfUIS3 can induce a consistent delay in patent parasitaemia across mouse strains and against chimeric parasites expressing PfUIS3 as well as wild type Pb; when this vaccine is combined with another partially protective regimen (ChAd63-MVA PfTRAP), complete protection is induced

Superior In Vitro Stimulation of Human CD8⁺ T-Cells by Whole Virus versus Split Virus Influenza Vaccines

<div><p>Pandemic and seasonal influenza viruses cause considerable morbidity and mortality in the general human population. Protection from severe disease may result from vaccines that activate antigen-presenting DC for effective stimulation of influenza-specific memory T cells. Special attention is paid to vaccine-induced CD8⁺ T-cell responses, because they are mainly directed against conserved internal influenza proteins thereby presumably mediating cross-protection against circulating seasonal as well as emerging pandemic virus strains. Our study showed that influenza whole virus vaccines of major seasonal A and B strains activated DC more efficiently than those of pandemic swine-origin H1N1 and pandemic-like avian H5N1 strains. In contrast, influenza split virus vaccines had a low ability to activate DC, regardless which strain was investigated. We also observed that whole virus vaccines stimulated virus-specific CD8⁺ memory T cells much stronger compared to split virus counterparts, whereas both vaccine formats activated CD4⁺ Th cell responses similarly. Moreover, our data showed that whole virus vaccine material is delivered into the cytosolic pathway of DC for effective activation of virus-specific CD8⁺ T cells. We conclude that vaccines against seasonal and pandemic (-like) influenza strains that aim to stimulate cross-reacting CD8⁺ T cells should include whole virus rather than split virus formulations.</p></div

Strain-specific influenza vaccines including type, used abbreviation, and status of regulatory approval.

a<p>results of clinical trials used to support license of A/H5N1/Vietnam/1203/2004.</p>b<p>licensed as trivalent seasonal vaccine.</p

Endolysosomal escape of whole virus vaccine material.

<p>Immature DC were incubated for 2(w/o) or with whole virus vaccines of pandemic-like avian strain A/H5N1-Vietnam (VNw) or seasonal strain B/Brisbane (BBw). Subsequently, samples were processed and analyzed by TEM using a Zeiss 912 Omega microscope operated at 120 kV. Pictures were taken at 5 increasing magnifications to allow better detection and tracking of virus material.</p

CD8⁺ T-cell reactivity to whole virus is superior compared to split virus preparations.

<p>CD4⁺ and CD8⁺ T cells purified from PBMC of healthy individuals were screened for IFN-γ ELISpot reactivity to autologous DC pre-loaded with 10 µg/mL of influenza whole virus and related split virus vaccine formulations. DC also received maturation cytokines during vaccine pulsing. (A) Representative data obtained from donor HD20 with 1×10⁵ CD4⁺ T cells (grey columns) or 1×10⁵ CD8⁺ T cells (black columns) plated per well are shown. (B, C) Reactivity to influenza whole virus and related split virus vaccines were measured in 10 randomly selected healthy individuals as described in (A). Box plot diagrams include IFN-γ ELISpot data from purified CD4⁺ (B) and CD8⁺ (C) T cells. Effective SFC were determined by subtraction of background spot numbers (w/o vaccine) from spot numbers induced by each individual vaccine. <i>P</i>-values were calculated by two-tailed paired-sample Wilcoxon signed-rank test.</p