• Construction and immunological evaluation of Salmonella vector vaccines that express HIV-1 envelope protein, gp120

      Fouts, Timothy Ray; Lewis, George K., Ph.D. (1994)
      Since the human immunodeficiency virus (HIV-1) is transmitted either parenterally or sexually, both mucosal and systemic immune responses may be required to provide protective immunity. Attenuated Salmonella vectors expressing heterologous antigen can stimulate responses in both compartments. To evaluate the utility of Salmonella vectors as a HIV-1 vector vaccine, a gene expression cassette encoding recombinant HIV-1 gp120 (rgp120) was integrated into the aroC locus of an attenuated vaccine strain of S. typhi. Immunoblot analysis using anti-gp120 monoclonal antibodies (mAb's) shows that the S. typhi strains containing the integrated cassette express a protein that is the appropriate size for non-glycosylated full-length gp120 (52 kDa). Immunoblot analysis also demonstrated that the recombinant S. typhi strains express the rgp120 as insoluble monomers and multimers. Antigen-capture ELISA, using antibodies specific for continuous epitopes on gp120, revealed that the S. typhi-expressed rgp120 is folded differently from a rgp120 derived from baculovirus that binds CD4. Immunization of BALB/c mice with S. typhi::rgp120 did not stimulate env-specific cytotoxic T lymphocytes (CTLs) or a significant rise in anti-gp120 antibody titers as compared to controls. Since mice may be a poor animal model for evaluating S. typhi vector vaccines, S. typhimurium strains were constructed that expressed rgp120 from a chromosomally integrated cassette (SL3261::120). To test if increased antigen expression potentiates immunogenicity, strains were constructed that express rgp120 from a multicopy asd stabilized plasmid (SL7207 pYA:120). Immunoblot analysis demonstrated that SL7207 pYA:120 expressed approximately 50 times more rgp120 than SL3261::120. Oral immunization of BALB/c mice with these strains did not stimulate an env-specific CTLs or a significant rise in anti-gp120 antibody titer as compared to controls. However, splenic T cells from SL7207 pYA:120 immunized mice proliferated upon restimulation with gp120 in vitro while splenocytes from SL3261::120 immunized mice did not. gp120 restimulated splenic T cells from SL7207 pYA:120 immune mice also produced IFN-{dollar}\gamma{dollar} but no IL-5. Three conclusions can be drawn from these results. First, Salmonella expresses rgp120 as a misfolded insoluble protein. Second, high level expression of rgp120 in Salmonella vectors is necessary to stimulate a gp120-specific immune response in mice. Third, Salmonella::rgp120 stimulates a gp120-specific Th1 response in mice. This report is the first to describe the construction of a Salmonella::rgp120 vector vaccine that is immunogenic in mice.
    • The Genomics and Epidemiology of Typhoid Fever in Samoa

      Sikorski, Michael Joseph; Levine, Myron M. (Myron Max), 1944-; Rasko, David A.; 0000-0002-3811-7285 (2022)
      Typhoid fever is a human host-restricted systemic infection caused by ingestion of fecally-contaminated food or water bearing Salmonella enterica serovar Typhi (S. Typhi). For decades, the Pacific Island nation of Samoa (population ~200,000) has faced unexplained, persistently endemic typhoid fever, despite improvements in water quality, sanitation, and economic development. Herein, epidemiologic analysis of surveillance data from 2008-2019 revealed that 53-193 blood culture-confirmed typhoid fever cases occurred annually in Samoa without seasonality. The greatest annual burden (number of cases/year) and incidence (cases/100,000 persons/year) occurred in primarily urban and peri-urban regions, where piped treated water reaches most households. Phylogenetic analyses of whole genome sequences (WGS) of 306 S. Typhi isolates from Samoa collected between 1983-2020 identified a dominant population of rare, Samoa-exclusive genotypes 3.5.3 and 3.5.4, which could be further divided into local sub-lineages. These Samoan genotypes are calculated to have most likely emerged in the 1970s with a shared common ancestry with other parent clade 3.5 isolates from South America, Southeast Asia, and Oceania. Furthermore, Samoan S. Typhi were reported susceptible to all clinically-relevant antibiotics, including those that are no longer effective in many other typhoid-endemic regions; indeed, resistance-conferring polymorphisms or genes were detected in <5% of isolates spanning decades of endemicity and despite suboptimal antibiotic prescribing practices. Finally, to elucidate patterns of transmission, point pattern spatial statistics and the high-resolution discerning power of WGS were integrated with detailed epidemiological data generated through household investigations of typhoid cases. Patterns identified were consistent with exposure within households in rural regions, dispersion of infections among a wider geographic area in urban regions, and transmission of genetically similar isolates among cases and asymptomatic shedders (carriers) identified through the household investigations. Notably, cases peaked during a period of increased population movement and dramatically decreased during two national lockdowns, indicating possible roles for population mobility and interaction in propagating typhoid fever. Together, these studies characterize the epidemiology and genomics of S. Typhi in Samoa, an endemic island setting in an understudied global region, and can directly assist the Samoa Ministry of Health and other similarly resourced Pacific Island public health programs in their typhoid control efforts.
    • In vivo characterization of the murine intranasal model for immunologic assessment of Salmonella typhi vaccine and live vector strains and use of this model to assess an S. typhi live vector strain expressing a Plasmodium falciparum antigen

      Pickett, Thames Emily; Levine, Myron M. (Myron Max), 1944- (1999)
      Attenuated Salmonella typhi live vector vaccine strains are highly immunogenic in mice following intranasal, but not orogastric inoculation. To elucidate the relationship between organs within which vaccine organisms are found and the induction of specific serum IgG antibodies, we examined the in vivo distribution of S. typhi vaccine strain CVD 908-htrA following intranasal administration. Vaccine organisms were cultured from the nasal-associated lymphoid tissue (NALT), lungs, and Peyer's patches two minutes after intranasal inoculation. Vaccine organisms persisted longer in NALT than in other organs. By decreasing the volume of intranasal inoculum containing 109 CFU (from a single 30 mul or 10 mul dose to four 2.5 mul doses given over the course of one hour), we were able to significantly reduce the number of vaccine organisms isolated from the lungs (p < 0.05), without reducing the number of vaccine organisms in NALT. Reducing the number of vaccine organisms in the lungs resulted in a significant decrease in the serum tetanus antitoxin response elicited by CVD 908-htrA expressing tetanus toxin fragment C under the control of the redox-responsive nir15 promoter. In contrast, a similar construct expressing tetanus toxin fragment C under control of the constitutive lpp promoter stimulated a strong serum IgG tetanus antitoxin response with both inoculation regimens. The data suggest that following intranasal inoculation, the NALT is a sufficient inductive site for elicitation of an immune response against both the live vector and heterologous antigen and, as occurs following oral inoculation of humans, attenuated S. typhi vaccine organisms elicit serum IgG responses. The murine intranasal model was used to assess the immunogenicity of CVD 908-htrA expressing an antigen from Plasmodium falciparum, the etiological agent of human malaria. A truncated version of the circumsporozoite protein (tCSP) was expressed as a translational fusion to tetanus toxin fragment C, under the control of either the nir15 or the ompC promoter. Mice inoculated with both constructs mounted appreciable serum IgG responses against S. typhi LPS; serum IgG responses against the fusion protein were weak or non-existent. However mice inoculated with both constructs exhibited specific lymphoproliferative responses against the live vector, tetanus toxoid and CSP.