James E. Galen

Conserved sequences in bacterial and viral sialidases.

The genes of the bacterial sialidases fromClostridium sordellii G12,C. perfringens A99,Salmonella typhimurium LT-2 andVibrio cholerae 395 sequenced so far were examined for homologies and were compared with sequences of viral sialidases.Each of the bacterial sialidases contains a short sequence of twelve amino-acids, which is repeated at four positions in the protein. All these sequences exhibit significant similarities. Comparing the repeated sequences of the four sialidases, five amino-acids were found to be highly conserved at defined positions: Ser-X-Asp-X-Gly-X-Thr-Trp. Additionally, most of the distances betweeen the four repeated regions are also conserved among the different sialidases. The conserved bacterial sequences show similarity with sialidases of influenza A H7N1 and H13N9.

Can a 'flawless' live vector vaccine strain be engineered?

The efficiency of any live bacterial vector vaccine hinges on its ability to present sufficient foreign antigen to the human immune system to initiate the desired protective immune response(s). However, synthesis of sufficient levels of heterologous antigen can result in an increase in metabolic burden with an accompanying decrease in the fitness of the live vector, which can ultimately lower desired immune responses to both live vector and heterologous antigen. Here, we explore the underlying mechanisms of metabolic load and propose ways of minimizing such burdens to enhance the fitness and immunogenicity of Salmonella-based live vector vaccines.

A murine model of intranasal immunization to assess the immunogenicity of attenuated Salmonella typhi live vector vaccines in stimulating serum antibody responses to expressed foreign antigens

The lack of a practical small animal model to study the immunogenicity of Salmonella typhi-based live vector vaccines expressing foreign antigens has seriously impeded the vaccine development process. For some foreign antigens, stimulation of serum IgG antibody is the desired, protective immune response. We administered to mice, by orogastric or intranasal (i.n.) routes, attenuated delta aroC delta aroD S. typhi CVD 908 carrying a plasmid encoding fragment C (fragC) of tetanus toxin fused to the eukaryotic cell receptor binding domain of diphtheria toxin (fragC-bDt), and monitored serum antibody. While orogastric inoculation of three doses was not immunogenic, i.n. immunization elicited high titers of serum IgG tetanus antitoxin, generating peak ELISA geometric mean titers (GMT) of 27024 and 35658 with 10(8) and 10(9) c.f.u. dosages, respectively; 10(9) c.f.u. i.n. of an delta aroA S. typhimurium live vector stimulated a peak antitoxin GMT of 376 405. Mice immunized with the S. typhi live vector were 100% protected against challenge with 100 50% lethal doses of tetanus toxin that rapidly killed all control mice. Intranasal immunization with two doses of S. typhi expressing unfused fragment C under control of an anaerobically-activated promoter derived from nirB stimulated significantly higher titers of serum neutralizing antitoxin than fused fragC-bDt controlled by the same promoter (GMT 0.10 AU ml-1 vs 0.01 AU ml-1, P = 0.0095). Two i.n. doses of S typhi encoding fragC under control of powerful constitutive promoter 1pp led to significantly higher peak serum neutralizing antitoxin titers than the otherwise identical construct utilizing the nirB promoter (peak GMT 0.72 AU ml-1 vs 0.10 AU ml-1, P = 0.022). The i.n. route of inoculation of mice may constitute a practical breakthrough that could expedite the development of some S. typhi-based live vector vaccines by allowing, for the first time, quantitative measurement of serum antibody responses to candidate constructs following i.n. mucosal immunization.

Attenuated Salmonella as live oral vaccines against typhoid fever and as live vectors.

Attenuated Salmonella typhi vaccine strain CVD 908, which harbors deletion mutations in aroC and aroD, has been shown to be well-tolerated and highly immunogenic, eliciting impressive serum antibody, mucosal IgA and cell-mediated immune responses. A further derivative prepared by introducing a deletion in htrA (which encodes a heat-shock protein that also has activity as a serine protease in CVD 908 (Chatfield et al., unpublished data) resulted in CVD 908-htrA. In phase 1 clinical trials, CVD 908-htrA appears very attractive as a live oral vaccine candidate. Both CVD 908 and CVD 908-htrA are useful as live vector vaccines to deliver foreign antigens to the immune system. Conditions that enhance the expression and immunogenicity of foreign antigens carried by CVD 908 and CVD 908-htrA are being investigated.

Identification by PCR of Non-typhoidal Salmonella enterica Serovars Associated with Invasive Infections among Febrile Patients in Mali

Background In sub-Saharan Africa, non-typhoidal Salmonella (NTS) are emerging as a prominent cause of invasive disease (bacteremia and focal infections such as meningitis) in infants and young children. Importantly, including data from Mali, three serovars, Salmonella enterica serovar Typhimurium, Salmonella Enteritidis and Salmonella Dublin, account for the majority of non-typhoidal Salmonella isolated from these patients. Methods We have extended a previously developed series of polymerase chain reactions (PCRs) based on O serogrouping and H typing to identify Salmonella Typhimurium and variants (mostly I 4,[5],12:i:-), Salmonella Enteritidis and Salmonella Dublin. We also designed primers to detect Salmonella Stanleyville, a serovar found in West Africa. Another PCR was used to differentiate diphasic Salmonella Typhimurium and monophasic Salmonella Typhimurium from other O serogroup B, H:i serovars. We used these PCRs to blind-test 327 Salmonella serogroup B and D isolates that were obtained from the blood cultures of febrile patients in Bamako, Mali. Principal Findings We have shown that when used in conjunction with our previously described O-serogrouping PCR, our PCRs are 100% sensitive and specific in identifying Salmonella Typhimurium and variants, Salmonella Enteritidis, Salmonella Dublin and Salmonella Stanleyville. When we attempted to differentiate 171 Salmonella Typhimurium (I 4,[ 5],12:i:1,2) strains from 52 monophasic Salmonella Typhimurium (I 4,[5],12:i:-) strains, we were able to correctly identify 170 of the Salmonella Typhimurium and 51 of the Salmonella I 4,[5],12:i:- strains. Conclusion We have described a simple yet effective PCR method to support surveillance of the incidence of invasive disease caused by NTS in developing countries.

Salmonella enterica Serovar Enteritidis Core O Polysaccharide Conjugated to H:g,m Flagellin as a Candidate Vaccine for Protection against Invasive Infection with S. Enteritidis

ABSTRACT Nontyphoidal Salmonella enterica serovars Enteritidis and Typhimurium are a common cause of gastroenteritis but also cause invasive infections and enteric fever in certain hosts (young children in sub-Saharan Africa, the elderly, and immunocompromised individuals). Salmonella O polysaccharides (OPS) and flagellar proteins are virulence factors and protective antigens. The surface polysaccharides of Salmonella are poorly immunogenic and do not confer immunologic memory, limitations overcome by covalently attaching them to carrier proteins. We conjugated core polysaccharide-OPS (COPS) of Salmonella Enteritidis lipopolysaccharide (LPS) to flagellin protein from the homologous strain. COPS and flagellin were purified from a genetically attenuated (ΔguaBA) “reagent strain” (derived from an isolate from a patient with clinical bacteremia) engineered for increased flagellin production (ΔclpPX). Conjugates were constructed by linking flagellin monomers or polymers at random COPS hydroxyls with various polysaccharide/protein ratios by 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) or at the 3-deoxy-d-manno-octulosonic acid (KDO) terminus by thioether chemistry. Mice immunized on days 0, 28, and 56 with COPS-flagellin conjugates mounted higher anti-LPS IgG levels than mice receiving unconjugated COPS and exhibited high antiflagellin IgG; anti-LPS and antiflagellin IgG levels increased following booster doses. Antibodies generated by COPS-flagellin conjugates mediated opsonophagocytosis of S. Enteritidis cells into mouse macrophages. Mice immunized with flagellin alone, COPS-CRM197, or COPS-flagellin conjugates were significantly protected from lethal challenge with wild-type S. Enteritidis (80 to 100% vaccine efficacy).

In Vivo Characterization of the Murine Intranasal Model for Assessing the Immunogenicity of Attenuated Salmonella enterica Serovar Typhi Strains as Live Mucosal Vaccines and as Live Vectors

ABSTRACT Attenuated Salmonella enterica serovar 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 immunoglobulin G (IgG) antibodies, we examined the in vivo distribution of serovar Typhi vaccine strain CVD 908-htrA following intranasal administration. Vaccine organisms were cultured from the nasal lymphoid tissue (NALT), lungs, and Peyers patches 2 min 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- or 10-μl dose to four 2.5-μl doses given over the course of 1 h), 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, 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 serovar Typhi vaccine organisms elicit serum IgG responses.

Salmonella enterica serovar Typhi Live Vector Vaccines Finally Come of Age

Attenuated Salmonella Typhi vaccine strains hold great promise as live vectors for presentation of foreign antigens from unrelated bacterial, viral and parasitic pathogens to the immune system. Although this approach has proved quite successful in experimental animal models for eliciting antigen‐specific mucosal, humoral and cellular responses, results have been disappointing for clinical trials carried out thus far. We hypothesize that the paucity of human responses to foreign antigens delivered by live vectors suggests that the strains and genetic approaches used to date have resulted in overattenuated vaccine strains with severely reduced immunogenicity. However, remarkable advances have now been made in the genetics of foreign antigen expression, understanding mechanisms of live vector immunity and refining immunization strategies. The time has now come for development of multivalent live vectors in which stable antigen expression and export is balanced with metabolic fitness to create highly immunogenic vaccines.

Adaptation of the Endogenous Salmonella enterica Serovar Typhi clyA-Encoded Hemolysin for Antigen Export Enhances the Immunogenicity of Anthrax Protective Antigen Domain 4 Expressed by the Attenuated Live-Vector Vaccine Strain CVD 908-htrA

ABSTRACT Bacterial live-vector vaccines aim to deliver foreign antigens to the immune system and induce protective immune responses, and surface-expressed or secreted antigens are generally more immunogenic than cytoplasmic constructs. We hypothesize that an optimum expression system will use an endogenous export system to avoid the need for large amounts of heterologous DNA encoding additional proteins. Here we describe the cryptic chromosomally encoded 34-kDa cytolysin A hemolysin of Salmonella enterica serovar Typhi (ClyA) as a novel export system for the expression of heterologous antigens in the supernatant of attenuated Salmonella serovar Typhi live-vector vaccine strains. We constructed a genetic fusion of ClyA to the reporter green fluorescent protein and showed that in Salmonella serovar Typhi CVD 908-htrA, the fusion protein retains biological activity in both domains and is exported into the supernatant of an exponentially growing live vector in the absence of detectable bacterial lysis. The utility of ClyA for enhancing the immunogenicity of an otherwise problematic antigen was demonstrated by engineering ClyA fused to the domain 4 (D4) moiety of Bacillus anthracis protective antigen (PA). A total of 11 of 15 mice immunized intranasally with Salmonella serovar Typhi exporting the protein fusion manifested fourfold or greater rises in serum anti-PA immunoglobulin G, compared with only 1 of 16 mice immunized with the live vector expressing cytoplasmic D4 (P = 0.0002). In addition, the induction of PA-specific gamma interferon and interleukin 5 responses was observed in splenocytes. This technology offers exceptional versatility for enhancing the immunogenicity of bacterial live-vector vaccines.

PCR Method To Identify Salmonella enterica Serovars Typhi, Paratyphi A, and Paratyphi B among Salmonella Isolates from the Blood of Patients with Clinical Enteric Fever

ABSTRACT PCR methodology was developed to identify Salmonella enterica serovars Typhi, Paratyphi A, and Paratyphi B. One multiplex PCR identifies serogroup D, A, and B and Vi-positive strains; another confirms flagellar antigen “d,” “a,” or “b.” Blinded testing of 664 Malian and Chilean Salmonella blood isolates demonstrated 100% sensitivity and specificity.