cGAMP loading enhances the immunogenicity of VLP vaccines

Abstract

23Cyclic GMP-AMP (cGAMP) is an immunostimulatory second messenger produced by cGAS 24 that activates STING. Soluble cGAMP acts as an adjuvant when administered with antigens. 25 cGAMP is also incorporated into enveloped virus particles during budding. We hypothesised 26 that inclusion of the adjuvant cGAMP within viral vaccine vectors would promote adaptive 27 immunity against vector antigens. We immunised mice with virus-like particles (VLPs) 28 containing the HIV-1 Gag protein and VSV-G. Inclusion of cGAMP within these VLPs 29 augmented splenic VLP-specific CD4 and CD8 T cell responses. It also increased VLP-and 30 VSV-G-specific serum antibody titres and enhanced in vitro virus neutralisation. The superior 31 antibody response was accompanied by increased numbers of T follicular helper cells in 32 draining lymph nodes. Vaccination with cGAMP-loaded VLPs containing haemagglutinin 33 induced high titres of influenza A virus neutralising antibodies and conferred protection 34 following subsequent influenza A virus challenge. Together, these results show that 35 incorporating cGAMP into VLPs enhances their immunogenicity, making cGAMP-VLPs an 36 attractive platform for novel vaccination strategies. 37 38 Running title: Immunogenicity of cGAMP-loaded VLP vaccines 39 GCs; and by supporting the generation of long-lived plasma cells and memory B cells (Crotty, 66 2019). In contrast, CD4 Tfr cells are involved in limiting GC reactions to prevent autoantibody 67 formation. Therefore, the Tfh/Tfr ratio is important for regulation of GC responses (Sage et al., 68 2013). 69 Virus-specific cytotoxic T cell (CTL) responses mediate clearance of infected cells to prevent 70 virus spread and eradicate infection. If sterilising immunity is not conferred by antibodies, 71 CTLs can make a key contribution to prophylactic vaccine efficacy (Hansen et al., 2011), and 72 they are critical for the control of persistent infection with viruses such as hepatitis B virus, 73 hepatitis C virus, cytomegalovirus and HIV (Panagioti et al., 2018). CTLs exert their activity 74 by triggering destruction of infected cells via release of perforins and granzymes; by ligation 75 of death-domain containing receptors and/or secretion of TNFa; and by producing "curative" 76 cytokines such as IFNg. Both the magnitude, i.e. the number of activated cells, and 77polyfunctionality of the T cell response, i.e. the capacity to mediate a breadth of effector 78 activities including production of multiple cytokines, are important determinants of CD8 T 79 cell-based vaccine efficacy (Panagioti et al., 2018). 80 Initiation of virus-specific CD4 and CD8 T cell responses requires presentation of viral 81antigens to naïve T cells by professional antigen-presenting cells (APCs), principally dendritic 82 cells (DCs). T cells need to receive three signals for activation: T cell receptor (TCR) triggering 83 by contact with peptide-major histocompatibility complexes (MHC) (signal 1); costimulatory 84 signals (signal 2); and inflammatory cytokines (signal 3) (Joffre et al.,...

Shaping neonatal immunization by tuning delivery of synergistic adjuvants via nanocarriers

“…To decipher the immunomodulatory effect of the admixture on germinal centers (GCs), we investigated the accumulation of T FH cells and GC B cells, plasmablasts and plasma cells by flow cytometry in the inguinal and popliteal draining lymph nodes (dLNs) on 12 days after the boost (Figure 5 & Figure S4). Consistent with previous observations that TLR8 signaling evokes T FH cell differentiation ex vivo 33,34 and that cGAMP with alum 13 or cGAMP loaded virus-like particles 35 induce T FH cells in dLNs of adult mice, cGAMP-PSs induced CD4 + T FH responses, as reflected by an increased in CD3 + CD4 + PD-1 + CXCR5 + T cells (Figure S4C). Despite being the inferior Th1 inducer in vivo, the co-encapsulated formulation triggered similar T FH responses when compared with the admixture formulation (Figure S1G).…”

Varicella‐Zoster virus ORF9 is an antagonist of the DNA sensor cGAS

“…The HEK293 cells used in this assay endogenously express STING. In response to cGAMP present in VLPs, these cells produce type I IFN, which we measured using a bioassay (Bridgeman et al, 2015;Chauveau et al, 2020). As expected, no type I IFN could be detected in response to infection with VLPs produced in the absence of cGAS (Figure 5D).…”

“…Transfection of HEK293T cells with plasmids encoding GFP-tagged HIV-1 gag and VSV-G results in the production of VSV-G pseudotyped, HIV-1 GFP-capsid containing virus-like particles (VLPs). Coexpression of cGAS during VLP production leads to the presence of cGAMP within the particles (Bridgeman et al, 2015;Chauveau et al, 2020). We hypothesised that the presence of ORF9 during VLP production blocks cGAS activation and thus prevents the presence of cGAMP in VLPs.…”

Varicella-Zoster Virus ORF9 Is an Antagonist of the DNA Sensor cGAS

“…The HEK293 333 cells used in this assay endogenously express STING. In response to cGAMP present 334in VLPs, these cells produce type I IFN, which we measured using a bioassay 335(Bridgeman et al, 2015;Chauveau et al, 2020). As expected, no type I IFN could be 336 detected in response to infection with VLPs produced in the absence of cGAS (Figure 3375D).…”

“…Transfection of HEK293T cells with 323 plasmids encoding GFP-tagged HIV-1 gag and VSV-G results in the production of 324 VSV-G pseudotyped, HIV-1 GFP-capsid containing virus-like particles (VLPs). Co-325 expression of cGAS during VLP production leads to the presence of cGAMP within the 326 particles (Bridgeman et al, 2015;Chauveau et al, 2020). We hypothesised that the 327 presence of ORF9 during VLP production blocks cGAS activation and thus prevents 328 the presence of cGAMP in VLPs.…”