Marcela F. Pasetti

Clinical trials of Shigella vaccines: two steps forward and one step back on a long, hard road

More than 50 years of research has yielded numerous Shigella vaccine candidates that have exemplified both the promise of vaccine-induced prevention of shigellosis and the impediments to developing a safe and effective vaccine for widespread use, a goal that has yet to be attained. This Review discusses the most advanced strategies for Shigella vaccine development, the immune responses that are elicited following disease or vaccination, the factors that have accelerated or impeded Shigella vaccine development and our ideas for the way forward.

Immunology of Gut Mucosal Vaccines

Summary:  Understanding the mechanisms underlying the induction of immunity in the gastrointestinal mucosa following oral immunization and the cross‐talk between mucosal and systemic immunity should expedite the development of vaccines to diminish the global burden caused by enteric pathogens. Identifying an immunological correlate of protection in the course of field trials of efficacy, animal models (when available), or human challenge studies is also invaluable. In industrialized country populations, live attenuated vaccines (e.g. polio, typhoid, and rotavirus) mimic natural infection and generate robust protective immune responses. In contrast, a major challenge is to understand and overcome the barriers responsible for the diminished immunogenicity and efficacy of the same enteric vaccines in underprivileged populations in developing countries. Success in developing vaccines against some enteric pathogens has heretofore been elusive (e.g. Shigella). Different types of oral vaccines can selectively or inclusively elicit mucosal secretory immunoglobulin A and serum immunoglobulin G antibodies and a variety of cell‐mediated immune responses. Areas of research that require acceleration include interaction between the gut innate immune system and the stimulation of adaptive immunity, development of safe yet effective mucosal adjuvants, better understanding of homing to the mucosa of immunologically relevant cells, and elicitation of mucosal immunologic memory. This review dissects the immune responses elicited in humans by enteric vaccines.

Adjuvanted Intranasal Norwalk Virus-Like Particle Vaccine Elicits Antibodies and Antibody-Secreting Cells That Express Homing Receptors for Mucosal and Peripheral Lymphoid Tissues

BACKGROUND Noroviruses cause significant morbidity and mortality from acute gastroenteritis in all age groups worldwide. METHODS We conducted 2 phase 1 double-blind, controlled studies of a virus-like particle (VLP) vaccine derived from norovirus GI.1 genotype adjuvanted with monophosphoryl lipid A (MPL) and the mucoadherent chitosan. Healthy subjects 18-49 years of age were randomized to 2 doses of intranasal Norwalk VLP vaccine or controls 21 days apart. Study 1 evaluated 5-, 15-, and 50-μg dosages of Norwalk antigen, and study 2 evaluated 50- and 100-μg dosages. Volunteers recorded symptoms for 7 days after dosing, and safety was followed up for 180 days. Blood samples were collected for serological profile, antibody secreting cells (ASCs), and analysis of ASC homing receptors. RESULTS The most common symptoms were nasal stuffiness, discharge, and sneezing. No vaccine-related serious adverse events occurred. Norwalk VLP-specific immunoglobulin G and immunoglobulin A antibodies increased 4.8- and 9.1-fold, respectively, for the 100-μg dosage level. All subjects tested who received the 50- or 100-μg vaccine dose developed immunoglobulin A ASCs. These cells expressed molecules associated with homing to mucosal and peripheral lymphoid tissues. CONCLUSIONS The intranasal monovalent adjuvanted Norwalk VLP vaccine was well tolerated and highly immunogenic and is a candidate for additional study.

Combination immunotherapy using adoptive T-cell transfer and tumor antigen vaccination on the basis of hTERT and survivin after ASCT for myeloma

In a phase 1/2 two-arm trial, 54 patients with myeloma received autografts followed by ex vivo anti-CD3/anti-CD28 costimulated autologous T cells at day 2 after transplantation. Study patients positive for human leukocyte antigen A2 (arm A, n = 28) also received pneumococcal conjugate vaccine immunizations before and after transplantation and a multipeptide tumor antigen vaccine derived from the human telomerase reverse transcriptase and the antiapoptotic protein survivin. Patients negative for human leukocyte antigen A2 (arm B, n = 26) received the pneumococcal conjugate vaccine only. Patients exhibited robust T-cell recoveries by day 14 with supraphysiologic T-cell counts accompanied by a sustained reduction in regulatory T cells. The median event-free survival (EFS) for all patients is 20 months (95% confidence interval, 14.6-24.7 months); the projected 3-year overall survival is 83%. A subset of patients in arm A (36%) developed immune responses to the tumor antigen vaccine by tetramer assays, but this cohort did not exhibit better EFS. Higher posttransplantation CD4(+) T-cell counts and a lower percentage of FOXP3(+) T cells were associated with improved EFS. Patients exhibited accelerated polyclonal immunoglobulin recovery compared with patients without T-cell transfers. Adoptive transfer of tumor antigen vaccine-primed and costimulated T cells leads to augmented and accelerated cellular and humoral immune reconstitution, including antitumor immunity, after autologous stem cell transplantation for myeloma. This study was registered at www.clinicaltrials.gov as NCT00499577.

Characterization of CD8+ Effector T Cell Responses in Volunteers Immunized with Salmonella enterica Serovar Typhi Strain Ty21a Typhoid Vaccine

Salmonella enterica serovar Typhi (S. typhi) strain Ty21a remains the only licensed attenuated typhoid vaccine. Despite years of research, the identity of the protective immunological mechanisms elicited by immunization with the Ty21a typhoid vaccine remains elusive. The present study was designed to characterize effector T cell responses in volunteers immunized with S. typhi strain Ty21a typhoid vaccine. We determined whether immunization with Ty21a induced specific CTL able to lyse S. typhi-infected cells and secrete IFN-γ, a key effector molecule against intracellular pathogens. We measured the functional activity of these CTL by a 51Cr-release assay using 8-day restimulated PBMC from Ty21a vaccinees as effector cells and S. Typhi-infected autologous PHA-activated PBMC as target cells. Most vaccinees exhibited consistently increased CD8-mediated lysis of targets by postimmunization PBMC when compared with preimmunization levels. We also developed an IFN-γ ELISPOT assay to quantify the frequency of IFN-γ spot-forming cells (SFC) in PBMC from Ty21a vaccinees using an ex vivo system. Significant increases in the frequency of IFN-γ SFC following immunization (mean ± SD, 393 ± 172; range 185–548 SFC/106 PBMC; p = 0.010), as compared with preimmunization levels, were observed. IFN-γ was secreted predominantly by CD8+ T cells. A strong correlation was recorded between the cytolytic activity of CTL lines and the frequency of IFN-γ SFC (r2 = 0.910, p < 0.001). In conclusion, this work constitutes the first evidence that immunization of volunteers with Ty21a elicits specific CD8+ CTL and provides an estimate of the frequency of CD8+ IFN-γ-secreting cells induced by vaccination.

Animal models paving the way for clinical trials of attenuated Salmonella enterica serovar Typhi live oral vaccines and live vectors

Attenuated Salmonella enterica serovar Typhi (S. Typhi) strains can serve as safe and effective oral vaccines to prevent typhoid fever and as live vectors to deliver foreign antigens to the immune system, either by the bacteria expressing antigens through prokaryotic expression plasmids or by delivering foreign genes carried on eukaryotic expression systems (DNA vaccines). The practical utility of such live vector vaccines relies on achieving a proper balance between minimizing the vaccines reactogenicity and maximizing its immunogenicity. To advance to clinical trials, vaccine candidates need to be pre-clinically evaluated in relevant animal models that attempt to predict what their safety and immunogenicity profile will be when administered to humans. Since S. Typhi is a human-restricted pathogen, a major obstacle that has impeded the progress of vaccine development has been the shortcomings of the animal models available to assess vaccine candidates. In this review, we summarize the usefulness of animal models in the assessment of the degree of attenuation and immunogenicity of novel attenuated S. Typhi strains as vaccine candidates for the prevention of typhoid fever and as live vectors in humans.

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.

Concomitant Induction of CD4+ and CD8+ T Cell Responses in Volunteers Immunized with Salmonella enterica Serovar Typhi Strain CVD 908-htrA

Type 1 cell-mediated immunity might play an important role in protection from typhoid fever. We evaluated whether immunization with Salmonella enterica serovar Typhi (S. Typhi) strain CVD 908-htrA (a ΔaroC ΔaroD ΔhtrA mutant), a leading live oral typhoid vaccine candidate, elicits specific CD4+ and CD8+ S. Typhi immune responses. Potent CTL responses and IFN-γ secretion by CD8+ T cells were detected following immunization with CVD 908-htrA in high (4.5 × 108 CFU) and low (5 × 107 CFU) dosages. S. Typhi-specific CTL were observed in six of eight vaccinees (four high and two low dose) after immunization. Mean increases in the frequency of IFN-γ spot-forming cells (SFC) in the presence of S. Typhi-infected targets were 221 ± 41 SFC/106 PBMC and 233 ± 87 SFC/106 PBMC, in the high and low dose groups, respectively. Strong CD4+ T cell responses were also observed. Increases in the IFN-γ production to soluble S. Typhi flagella (STF) occurred in 82 and 38% of the volunteers who received the high and low doses, respectively. Robust correlations were observed between volunteers that responded with IFN-γ SFC to stimulation with S. Typhi-infected cells and IFN-γ released in response to stimulation with STF Ags (r = 0.822, p < 0.001) and between CTL and IFN-γ production to STF (r = 0.818, p = 0.013). These data demonstrating the concomitant induction of both CD4- and CD8-mediated CMI are consistent with a significant role for type 1 immunity in controlling typhoid infection and support the continuing evaluation of CVD 908-htrA as a typhoid vaccine candidate.

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.