3c). The results were obtained in two independent groups of BLT-NSG mice engrafted with HLA-A2+ thymus and liver. Together our data indicate that T cells obtained from selleck kinase inhibitor BLT-NSG mice during acute infection and in the memory phase secrete cytokines in response to stimulation with multiple DENV peptide pools as well as known HLA-A2-restricted DENV peptides. We next assessed the generation of DENV-2-specific antibodies in DENV-infected BLT-NSG mice by sandwich
ELISA. Sera from DENV-2 NGC-infected BLT-NSG mice had significantly higher IgM antibody responses against the DENV-2 envelope protein compared with responses detected in HLA-A2-transgenic NSG mice engrafted with human cord blood HSC (Fig. 4a) and previously published data in HLA-A2 cord blood Ruxolitinib mouse HSC-engrafted NSG mice.14 High IgM responses
were consistently validated in the sera of mice up to 8 weeks post-infection (Fig. 4b). Little or no DENV-specific IgG was detected even 8 weeks post-infection with DENV-2 NGC (Fig. 4b). We assessed whether multiple immunizations with DENV-2 NGC would enhance antibody responses and found a modest increase in IgM antibodies in the sera of mice that were infected more than once with DENV (Fig. 4c). No IgG responses were detected in the sera of mice immunized multiple times (data not shown). To determine whether the strain and dose of DENV influenced antibody responses, we infected mice with increasing doses of DENV-2 S16803 (a live-attenuated vaccine strain) (Fig. 4d). We found similar IgM antibody responses in the sera of mice infected with DENV-2 NGC and DENV S16803. Irrespective of the inoculation dose IgM responses were similar and in all cases we detected
low DENV-specific IgG responses. Our data indicate that IgM antibodies, which are neither viral strain-dependent nor dose-dependent, are the predominant isotype produced in response to dengue viral infection in BLT-NSG mice. Experiments were conducted next to determine whether splenic B cells from BLT-NSG mice were able to secrete DENV-specific antibodies. We used culture supernatants from stimulated splenocytes as a source of DENV-specific antibodies. We were able to detect antibodies in the supernatants of immune but not naive splenocytes from BLT-NSG mice that bound an inactivated lysate of DENV-2 and the DENV-2 Coproporphyrinogen III oxidase E protein (Fig. 5a). We next tested the neutralizing activity of DENV-2-specific antibodies generated by B cells in infected mice. We found that supernatants obtained from stimulated splenocytes of DENV-2-infected mice inhibited DENV-2 infection of Vero cells whereas supernatants obtained from stimulated naive splenocytes were unable to reduce infection (Fig. 5b). A summary of DENV-specific neutralizing activity (41–97% neutralization at 1 : 5 dilution) (n = 6) in supernatants obtained from splenocytes of infected mice is shown (Fig. 5c).