Nipah virus vaccines evaluated in pigs as a 'One Health' approach to protect public health

Abstract

Nipah virus (NiV) causes a severe neurological disease in humans. The first NiV outbreak, in Malaysia, involved pig-to-human transmission, that resulted in significant economic losses to the local pig industry. Despite the risk NiV poses to pig-dense regions, no licensed vaccines exist. This study therefore assessed three NiV vaccine candidates in pigs: (1) adjuvanted soluble NiV (s)G protein, (2) adjuvanted pre-fusion stabilised NiV (mcs)F protein, and (3) adenoviral vectored NiV G (ChAdOx1 NiV G). NiV sG induced the strongest neutralising antibody response, NiV mcsF induced antibodies best able to neutralise cell-cell fusion, whereas ChAdOx1 NiV G elicited CD8⁺ T-cell responses. Despite differences in immunogenicity, prime-boost immunisation with all candidates conferred a high degree of protection against NiV infection. Follow-up studies demonstrated longevity of immune responses and broadly comparable immune responses in Bangladeshi pigs under field conditions. These studies provide a platform for developing a NiV vaccine for pigs.

Fig. 1. Immunisation of mice with NiV sG, ChAdOx1 NiV G or NiV mcsF induces varied antibody and T-cell responses.

Mice were immunised on day 0 and 21 by intramuscular inoculation of 5 µg NiV sG, NiV mcsF, or HeV sG proteins in adjuvant, or 1 × 10⁸ IU ChAdOx1 NiV G. A NiV mcsF and sG binding antibody titres (EPT) in serum on day 21 and 42; B Virus neutralising titres in day 21 and 42 serum as assessed by NiV_M VNT; C, D CD8⁺ and CD4⁺ T-cell cytokine responses after stimulation of splenocytes with NiV G peptides, respectively; E, F CD8⁺ and CD4⁺ T-cell cytokine responses after stimulation of splenocytes with NiV F peptides, respectively. Splenocytes from all groups were restimulated with NiV G peptides, whereas splenocytes from the NiV sG, NiV mcsF and adjuvant only groups were restimulated with NiV F peptides. Datapoints represent individual mice, with the bars showing the group mean and error bars represent the standard deviation (SD). Significant differences were determined using two-way ANOVA and signified with the following letter: a—significant difference to adjuvant; b—significant difference to NiV sG; c—significant difference to ChAdOx1 NiV G; d—significant difference to NiV mcsF; e—significant difference to HeV sG.

Fig. 2. Immunisation of pigs with NiV sG, ChAdOx1 NiV G, NiV mcsF similarly induces varied antibody and T-cell responses.

Pigs were immunised on day 0 and 21 by intramuscular inoculation of 100 µg NiV sG, NiV mcsF, or HeV sG proteins in adjuvant, or 1 × 10⁹ IU ChAdOx1 NiV G. A NiV mcsF and sG binding antibody titres (EPT) on day 21 and 42; B Virus neutralising titres as assessed by NiV_M VNT; C Inhibition of NiV glycoprotein-mediated cell-cell fusion evaluated using day 42 sera; D, E CD8⁺ and CD4⁺ T-cell cytokine responses after stimulation of PBMC with NiV G peptide pool, respectively; F, G CD8⁺ and CD4⁺ T-cell cytokine responses after stimulation of PBMC with NiV F peptide pool, respectively. H, I IFN-γ ELISpot assay to assess PBMC responses to NiV G and F peptide stimulation, respectively. For A and C, datapoints represent individual pigs, with the bars showing the group mean and error bars represent the SD. For the other panels, datapoints represent the group mean and error bars represent the SD. Significant differences were determined using two-way ANOVA and signified with the following letter: a—significant difference to adjuvant; b—significant difference to NiV sG; c—significant difference from ChAdOx1 NiV G; d—significant difference to NiV mcsF; e—significant difference to HeV sG.

Fig. 3. NiV vaccine candidates are protective in pigs following a prime-boost immunisation regimen.

Pigs were immunised on day 0 and 21 by intramuscular inoculation of 100 µg NiV sG or NiV mcsF proteins in adjuvant, or 1 × 10⁹ IU ChAdOx1 NiV G. On day 42, all pigs were challenged by oronasal inoculation with 1 × 10⁵ PFU NiV_M. A Neutralising antibody titres as assessed by NiV_M VNT; B, C Viral loads in nasal and oral swabs detected post-challenge by RT-qPCR, respectively. D, E Level of infectious NiV from nasal and oral swabs detected post-challenge by virus isolation, respectively. F, G Viral loads from tissue samples at 6 days post-challenge detected by RT-qPCR and virus isolation, respectively. Postmortem tissues collected: PSLN prescapular lymph node, RPLN retropharyngeal lymph node, SMLN submandibular lymph node, TBLN tracheobronchial lymph nodes, Olf bulb olfactory bulb, Trig ganglion trigeminal ganglion. Significant differences were determined using two-way ANOVA and signified with the following letter: a—significant difference to unvaccinated; b—significant difference to NiV sG; c—significant difference from ChAdOx1 NiV G; d—significant difference to NiV mcsF. NS not significant.

Fig. 4. NiV vaccine candidates are not effective in pigs following a prime-only immunisation regimen.

Pigs were immunised on day 0 by intramuscular inoculation of 100 µg NiV sG or NiV mcsF proteins in adjuvant, or 1 × 10⁹ IU ChAdOx1 NiV G. On day 21, all pigs were challenged by oronasal inoculation of 1 × 10⁵ PFU NiV_M. A Neutralising antibody titres as assessed by NiV_M VNT; B, C Viral loads in nasal and oral swabs detected post-challenge by RT-qPCR, respectively. D, E Level of infectious NiV from nasal and oral swabs detected post-challenge by virus isolation, respectively. F, G Viral loads from tissue samples at 6 days post-challenge detected by RT-qPCR and virus isolation, respectively. Postmortem tissues collected: PSLN prescapular lymph node, RPLN retropharyngeal lymph node, SMLN submandibular lymph node, TBLN tracheobronchial lymph nodes, Olf bulb olfactory bulb, Trig ganglion trigeminal ganglion. Significant differences were determined using two-way ANOVA and signified with the following letter: a—significant difference to unvaccinated.

Fig. 5. Comparison of the immunogenicity of NiV sG, ChAdOx1 NiV G, and NiV mcsF following prime only and prime-boost immunisation regimens.

All pigs were immunised on day 0 and 3 out of the 6 groups were boosted on day 21 by intramuscular inoculation of 100 µg NiV sG or NiV mcsF proteins in adjuvant, or 1 × 10⁹ IU ChAdOx1 NiV G. A NiV sG and mcsF binding antibody titres (EPT) on day 21, 42 and 112; B Virus neutralising titres as assessed by NiV_M VNT; C Inhibition of NiV glycoprotein-mediated cell-cell fusion evaluated using day 112 sera; D, E IFN-γ ELISpot assay to assess PBMC responses to NiV G and F peptide stimulation, respectively. For A and C, datapoints represent individual pigs, with the bars showing the group mean and error bars represent the SD. For the other panels, datapoints represent the group mean and error bars represent the SD. Significant differences were determined using two-way ANOVA and signified with the following letter: b—significant difference to NiV sG prime-boost; c—significant difference to ChAdOx1 NiV G prime-boost; d—significant difference to NiV mcsF prime-boost; f—significant difference to NiV sG prime only; g—significant difference to ChAdOX1 NiV G prime only; h—significant difference to NiV mcsF prime only.

Fig. 6. Immunogenicity of NiV sG, ChAdOx1 NiV G and NiV mcsF in indigenous backyard pigs under field conditions in the ‘Nipah belt’ of Bangladesh.

Pigs were immunised on day 0 and 21 by intramuscular inoculation of 100 µg NiV sG or NiV mcsF proteins in adjuvant, or 1 × 10⁹ IU ChAdOx1 NiV G. A NiV mcsF and sG binding antibody titres (EPT) on day 42; B, C Virus neutralising titres as assessed by NiV_M VNT and pseudoVNT, respectively; D Inhibition of NiV glycoprotein-mediated cell-cell fusion evaluated using day 42 sera. Significant differences were determined using one-way ANOVA (A, B and D) or two-way ANOVA (C) and signified with the following letter: b—significant difference to NiV sG; c—significant difference to ChAdOx1 NiV G; d—significant difference to NiV mcsF.

Fig. 7. Schematic summary of the evaluation of NiV vaccine candidates in pigs.

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