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. 2020 Jan 22;88(2):e00573-19.
doi: 10.1128/IAI.00573-19. Print 2020 Jan 22.

Preclinical Development and Assessment of Viral Vectors Expressing a Fusion Antigen of Plasmodium falciparum LSA1 and LSAP2 for Efficacy against Liver-Stage Malaria

Benedict R Halbroth  1 , Sarah Sebastian  1 , Ahmed M Salman  1   2 , Marta Ulaszewska  1 , Anita Gola  1 , Rhea J Longley  1 , Chris J Janse  2 , Shahid M Khan  2 , Adrian V S Hill  1 , Alexandra J Spencer  3
Affiliations

Preclinical Development and Assessment of Viral Vectors Expressing a Fusion Antigen of Plasmodium falciparum LSA1 and LSAP2 for Efficacy against Liver-Stage Malaria

Benedict R Halbroth et al. Infect Immun. .

Abstract

Despite promising progress in malaria vaccine development in recent years, an efficacious subunit vaccine against Plasmodium falciparum remains to be licensed and deployed. Cell-mediated protection from liver-stage malaria relies on a sufficient number of antigen-specific T cells reaching the liver during the time that parasites are present. A single vaccine expressing two antigens could potentially increase both the size and breadth of the antigen-specific response while halving vaccine production costs. In this study, we investigated combining two liver-stage antigens, P. falciparum LSA1 (PfLSA1) and PfLSAP2, and investigated the induction of protective efficacy by coadministration of single-antigen vectors or vaccination with dual-antigen vectors, using simian adenovirus and modified vaccinia virus Ankara vectors. The efficacy of these vaccines was assessed in mouse malaria challenge models using chimeric P. berghei parasites expressing the relevant P. falciparum antigens and challenging mice at the peak of the T cell response. Vaccination with a combination of the single-antigen vectors expressing PfLSA1 or PfLSAP2 was shown to improve protective efficacy compared to vaccination with each single-antigen vector alone. Vaccination with dual-antigen vectors expressing both PfLSA1 and PfLSAP2 resulted in responses to both antigens, particularly in outbred mice, and most importantly, the efficacy was equivalent to that of vaccination with a mixture of single-antigen vectors. Based on these promising data, dual-antigen vectors expressing PfLSA1 and PfLSAP2 will now proceed to manufacturing and clinical assessment under good manufacturing practice (GMP) guidelines.

Keywords: T cells; liver stage; malaria; vaccines.

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Figures

FIG 1
FIG 1
Immunogenicity and efficacy of coadministration of PfTRAP and PfLSA1. (A and B) BALB/c mice (6 per group) were vaccinated with 108 IU ChAd63, followed 7 weeks later with a 106-IU MVA boost of each vaccine expressing PfTRAP (circles), PfLSA1 (squares), both vaccines administered in separate legs (open triangles), or both vaccines mixed (diamonds). One week post-MVA boost (week 7), a blood sample was taken, and PBMCs were analyzed by ICS after stimulation with a PfTRAP or PfLSA1 peptide pool. Ten days after MVA boost, the mice were challenged with 1,000 chimeric P. berghei sporozoites expressing P. falciparum PfTRAP and PfLSA1 and monitored for development of blood-stage malaria. The graphs represent the frequency of blood CD4+ IFN-γ+ (A) or CD8+ IFN-γ+ (B) T cells. (C) Time to reach 1% parasitemia plotted on a Kaplan-Meier survival curve. The data points indicate individual mice, and the horizontal lines show the median response per group.
FIG 2
FIG 2
Immunogenicity and efficacy of coadministration of PfTRAP and PfLSAP2. (A and B) BALB/c mice (6 per group) were vaccinated with 108 IU ChAd63 followed 7 weeks later with a 106-IU MVA boost of each vaccine expressing PfTRAP, PfLSAP2, both vaccines administered in separate legs, or both vaccines mixed. One week post-MVA boost (week 7), a blood sample was taken, and PBMCs were analyzed by ICS after stimulation with a PfTRAP or PfLSAP2 peptide pool. Ten days after MVA boost, the mice were challenged with 1,000 chimeric P. berghei sporozoites expressing PfTRAP and PfLSAP2 and monitored for development of blood-stage malaria. The graphs represent the frequency of blood CD4+ IFN-γ+ (A) or CD8+ IFN-γ+ (B) T cells. (C) Time to reach 1% parasitemia plotted on a Kaplan-Meier survival curve. The data points indicate individual mice, and the horizontal lines show the median response per group.
FIG 3
FIG 3
Immunogenicity and efficacy of coadministration of PfLSA1 and PfLSAP2. (A to C) BALB/c mice (8 per group) were vaccinated with 108 IU ChAd63 expressing PfLSA1, PfLSAP2, or both vaccines mixed (Mix), resulting in a total virus dose of 2 × 108 IU. A blood sample was taken on day 14, and PBMCs were analyzed by ICS after stimulation with a PfLSA1 or PfLSAP2 peptide pool. At day 17 postvaccination, the mice were challenged with 1,000 chimeric P. berghei sporozoites expressing PfLSA1 and PfLSAP2 and monitored for development of blood-stage malaria. The graphs represent the frequency of blood CD4+ IFN-γ+ (A) or CD8+ IFN-γ+ (B) T cells or summed CD4+ IFN-γ+ or CD8+ IFN-γ+ T cell responses (C). The bars represent median response; error bars indicate standard error of the mean. (D) Time to reach 1% parasitemia plotted on a Kaplan-Meier survival curve. The data points indicate individual mice, and the horizontal lines show the median response per group.
FIG 4
FIG 4
Immunogenicity of dual-antigen-expressing ChAdOx1 in inbred and outbred mice. (A) BALB/c mice (5 per group) were immunized with 108 IU of each ChAdOx1 vector expressing either PfLSA1, PfLSAP2, both vectors administered into separate legs (Separate), both vectors premixed and injected into both legs (Mixed), or dual-antigen-expressing vector (Dual Ag ×1) or 2 × 108 IU of the dual-antigen-expressing vector (Dual Ag ×2). T cell responses to PfLSA1 or PfLSAP2 peptide pools were analyzed by ICS. The percentages of blood CD4+ and CD8+ T cells positive for IFN-γ are shown. The single points represent individual mice; the horizontal lines denote the median response per group. To determine if there was a difference between the total antigen-specific response, CD4+ or CD8+ IFN-γ+ responses to each antigen were summed, and the data were analyzed with a two-way repeated-measures ANOVA with post hoc positive effect to determine the effect of vaccine for each T cell subset. The P values denote the levels of significance observed. The bars represent median response; error bars indicate standard error of the mean. (B) CD1 mice (6 per group) were immunized with 108 IU ChAdOx1 vectors as for panel A, with spleens harvested 2 weeks later. T cell responses to PfLSA1 and PfLSAP2 peptide pools were analyzed by ICS. The percentages of CD4+ and CD8+ T cells positive for IFN-γ are shown. To determine if there was a difference between the total antigen-specific response, CD4+ or CD8+ IFN-γ+ responses to each antigen were summed, and the data were analyzed with a two-way repeated-measures ANOVA, but no significant effect was observed for either T cell subset or vaccine.
FIG 5
FIG 5
Immunogenicity and efficacy of dual-antigen-expressing vectors in BALB/c mice following prime-boost vaccination. (A to C) BALB/c mice (6 per group) were immunized with 108 IU of ChAdOx1 (the Mix 2× group received 2 × 108 IU total virus) and boosted 6 weeks later with 107 PFU MVA (the Mix 2× group received 2 × 107 PFU total virus), with antigen inserts as indicated on the x axes. In ChAdOx1 vectors, the antigen was fused to the adjuvant sharkTM/Ii; in MVA vectors, the antigen was fused to tPA. A blood sample was taken in week 7 and analyzed by ICS after stimulation with a PfLSA1 (A) or PfLSAP2 (B) peptide pool. The graphs represent the frequency of PfLSA1-specific (A) or PfLSAP2 (B) CD4+ IFN-γ+ or CD8+ IFN-γ+ T cells or the summed CD4+ I FN-γ+ or CD8+ IFN-γ+ T cell responses (C). Mice were challenged with 1,000 chimeric P. berghei sporozoites expressing PfLSA1 and PfLSAP2 and monitored for development of blood-stage malaria. The bars represent median response; error bars indicate standard error of the mean. (D) Time to reach 1% parasitemia plotted on a Kaplan-Meier survival curve. All the groups showed significant increases in survival compared to naive controls above the Bonferroni-corrected threshold (P < 0.003), but no significance (n.s.) between vaccinated groups was observed.
FIG 6
FIG 6
Immunogenicities and efficacies of dual-antigen-expressing vectors in outbred mice. (A to D) CD-1 mice (10 per group) were immunized with 108 IU of ChAdOx1 (The Mix 2× group received 2 × 108 IU total virus) and boosted 6 weeks later with 107 PFU MVA (the Mix 2× group received 2 × 107 PFU total virus), with antigen inserts as indicated on the x axes. In ChAdOx1 vectors, the antigen was fused to the adjuvant sharkTM/Ii; in MVA vectors, the antigen was fused to tPA (sharkTM/Ii → tPA). A blood sample was taken in week 7 and analyzed by ICS after stimulation with a PfLSA1 (A) or PfLSAP2 (B) peptide pool. The graphs represent the frequency of CD4+ IFN-γ+ (A) or CD8+ IFN-γ+ (B) PfLSA1 or PfLSAP2 T cells, summed CD4+ IFN-γ+ or CD8+ IFN-γ+ T cell response (C), or the percentage of CD4+ IFN-γ+ or CD8+ IFN-γ+ T cells specific for either PfLSA1 or PfLSAP2 (D). The mice were challenged with 1,000 chimeric P. berghei sporozoites expressing PfLSA1 and PfLSAP2 and monitored for development of blood-stage malaria. (E) Time to 1% parasitemia plotted on a Kaplan-Meier survival curve. Only PfLSAP2 and PfLSA1-LSAP2 showed a significant increase in survival compared to naive controls above the Bonferroni-corrected threshold (P < 0.003), but no significance between vaccinated groups was observed. The error bars indicate standard deviations.
FIG 7
FIG 7
Prime-target immunization improves the efficacy of the dual LSA1-LSAP2-expressing vectors. (A) BALB/c mice (6 per group) were immunized with 108 IU of ChAdOx1.LSA1-LSAP2 (i.m.), boosted 2 weeks later with 106 PFU MVA.LSA1-LSAP2 (i.m.), and targeted a further 2 weeks later with 107 PFU MVA.LSA1-LSAP2 (i.v. or i.m.). Alternatively, mice were primed with 108 IU of ChAdOx1.LSA1-LSAP2 (i.m.) and targeted 2 weeks later with 107 PFU MVA.LSA1-LSAP2 (i.v. or i.m.). The mice were challenged with 1,000 chimeric P. berghei sporozoites expressing PfLSA1 and PfLSAP2 3 weeks after the targeting immunization and monitored for development of blood-stage malaria. The time to reach 1% parasitemia is plotted on a Kaplan-Meier survival curve. All the groups showed significant increases in survival compared to naive controls above the Bonferroni-corrected threshold (P < 0.008), with a significant difference observed between Adim-MVAim and Adim-MVAiv. (B) CD-1 mice (10 per group) were immunized with 108 IU of ChAdOx1.LSA1-LSAP2 (i.m.), boosted 4 weeks later with 106 PFU MVA.LSA1-LSAP2 (i.m.), and targeted a further 2 weeks later with 109 IU ChAdOx1.LSA1-LSAP2 (i.m.). The mice were challenged with 1,000 chimeric P. berghei sporozoites expressing PfLSA1 and PfLSAP2 3 weeks after the final immunization and monitored for development of blood-stage malaria. The time to reach 1% parasitemia is plotted on a Kaplan-Meier survival curve. Both groups showed significant increases in survival compared to naive controls above the Bonferroni-corrected threshold (P < 0.017), but a statistically significant difference between targeting of mice with Ad administered i.v. or i.m. was not observed.
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