Browsing by Author "Margolin, Emmanuel"
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- ItemOpen AccessAssessment of an LSDV-Vectored Vaccine for Heterologous Prime-Boost Immunizations against HIV(2021-11-05) Chapman, Ros; van Diepen, Michiel; Douglass, Nicola; Galant, Shireen; Jaffer, Mohamed; Margolin, Emmanuel; Ximba, Phindile; Hermanus, Tandile; Moore, Penny L; Williamson, Anna-LiseThe modest protective effects of the RV144 HIV-1 vaccine trial have prompted the further exploration of improved poxvirus vector systems that can yield better immune responses and protection. In this study, a recombinant lumpy skin disease virus (LSDV) expressing HIV-1 CAP256.SU gp150 (Env) and a subtype C mosaic Gag was constructed (LSDVGC5) and compared to the equivalent recombinant modified vaccinia Ankara (MVAGC5). In vitro characterization confirmed that cells infected with recombinant LSDV produced Gag virus-like particles containing Env, and that Env expressed on the surface of the cells infected with LSDV was in a native-like conformation. This candidate HIV-1 vaccine (L) was tested in a rabbit model using different heterologous vaccination regimens, in combination with DNA (D) and MVA (M) vectors expressing the equivalent HIV-1 antigens. The four different vaccination regimens (DDMMLL, DDMLML, DDLMLM, and DDLLMM) all elicited high titers of binding and Tier 1A neutralizing antibodies (NAbs), and some regimens induced Tier 1B NAbs. Furthermore, two rabbits in the DDLMLM group developed low levels of autologous Tier 2 NAbs. The humoral immune responses elicited against HIV-1 Env by the recombinant LSDVGC5 were comparable to those induced by MVAGC5.
- ItemOpen AccessDevelopment and Characterization of a Modified Vaccinia Ankara Vaccine Candidate Expressing the SARS-CoV-2 Spike Glycoprotein(2022) Khumalo, Fezokuhle Ncedile; Margolin, Emmanuel; Williamson, Anna-Lise; Douglass, NicolaCoronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2, SARS-CoV-2. Given the ongoing COVID-19 pandemic and the continued evolution of the virus to escape host immunity, new vaccines and refinement of first-generation vaccines to improve protection against SARS-CoV-2 variants of concern is vital. In Africa, the cost of vaccine manufacturing as well as the scarcity in resources for storage and distribution have all contributed to the inequitable access to vaccines and heavy reliance on donations. Modified Vaccinia Virus Ankara (MVA) is a low-cost production vector platform which is suitable in this context. This project falls into a bigger study where our group compared different vector platforms, including MVA. The project serves as a proof of concept that this platform can be used to produce vaccines encompassing different variants of SARS-CoV-2 as they emerge. The most recent variant, Omicron, has proven to be highly immune evasive and demonstrates this need well. As the virus mutates, the variants of concern each present with differing characteristics and subsequently differ in immunogenicity and pathogenicity. Sub-Saharan Africa has been ransacked by the pandemic, resulting in loss of lives and livelihoods; the effects of which will undoubtedly be felt for decades to come. This study had two aims: 1. The development of a candidate vaccine, MVA-SARS-CoV2-S∆TM by using the widely used MVA platform and poxvirus recombinant vaccine strategies used in our research group, 2. The testing of this vaccine's immunogenicity in mice. The MVA-based vaccine was constructed by infection of BHK21 cells with wildtype MVA, and transfection with transfer vector pMVA-FNK2. The transfer vector contains a truncated form of the SARS-CoV-2 spike glycoprotein gene, the vaccinia virus host-range gene K1L and reporter gene eGFP, flanked by gene sequences to allow homologous recombination into MVA. The presence of the K1L gene in the recombinant virus allowed for selection by passaging in RK13 cells, which were not permissive for the parent MVA. Following the potentially successful isolation, the recombinant MVA-SARS-CoV2-S∆TM was validated and characterized by PCR, Sanger sequencing, Western blot analysis and immunofluorescence, which all confirmed the presence and expression of the spike protein. Large scale propagation of the vaccine was done in RK13 cells, and the stock was titrated to yield a titre of 3.9 x 106 ffu/ml. Balb/c mice were inoculated three times with MVA-SARS-CoV2-S∆TM at a dose of 3 x 105 to assess immunogenicity of the vaccine. Results from the immunogenicity assessments demonstrated that the vaccine induced T-cell responses shown by an enzyme-linked immunosorbent spot (ELISpot) assay. An enzyme-linked immunosorbent assay (ELISA) confirmed the ability of MVA-SARSCoV2-S∆TM to induce increasing titres of binding antibodies in mice over a period of 56 days. However, the vaccine did not induce neutralizing antibodies against a matched SARS-CoV-2 pseudovirus, highlighting the need for further refinement of the vaccine. In conclusion, recombinant MVA expressing a truncated SARS-CoV-2 spike protein was successfully constructed and tested for immunogenicity in mice. The candidate vaccine induced good T-cell responses and binding antibodies, but not neutralizing antibodies. This study provides additional evidence that MVA can be used as a platform for a SARS-CoV-2 vaccine, as has been previously demonstrated by others, and this could potentially be adapted for emerging variants of concern. This work also allows for the direct comparison of the MVA platform to other platforms employed by our group (DNA and plant-based subunit) as SARS-CoV-2 vaccines.
- ItemOpen AccessLSDV-Vectored SARS-CoV-2 S and N Vaccine Protects against Severe Clinical Disease in Hamsters(2023-06-21) de Moor, Warren R. J.; Williamson, Anna-Lise; Schäfer, Georgia; Douglass, Nicola; Gers, Sophette; Sutherland, Andrew D.; Blumenthal, Melissa J.; Margolin, Emmanuel; Shaw, Megan L.; Preiser, Wolfgang; Chapman, RosamundThe SARS-CoV-2 pandemic demonstrated the need for potent and broad-spectrum vaccines. This study reports the development and testing of a lumpy skin disease virus (LSDV)-vectored vaccine against SARS-CoV-2, utilizing stabilized spike and conserved nucleocapsid proteins as antigens to develop robust immunogenicity. Construction of the vaccine (LSDV-SARS2-S,N) was confirmed by polymerase chain reaction (PCR) amplification and sequencing. In vitro characterization confirmed that cells infected with LSDV-SARS2-S,N expressed SARS-CoV-2 spike and nucleocapsid protein. In BALB/c mice, the vaccine elicited high magnitude IFN-γ ELISpot responses (spike: 2808 SFU/106 splenocytes) and neutralizing antibodies (ID50 = 6552). Testing in hamsters, which emulate human COVID-19 disease progression, showed the development of high titers of neutralizing antibodies against the Wuhan and Delta SARS-CoV-2 variants (Wuhan ID50 = 2905; Delta ID50 = 4648). Additionally, hamsters vaccinated with LSDV-SARS2-S,N displayed significantly less weight loss, lung damage, and reduced viral RNA copies following SARS-CoV-2 infection with the Delta variant as compared to controls, demonstrating protection against disease. These data demonstrate that LSDV-vectored vaccines display promise as an effective SARS-CoV-2 vaccine and as a potential vaccine platform for communicable diseases in humans and animals. Further efficacy testing and immune response analysis, particularly in non-human primates, are warranted.