Outer membrane vesicle vaccines.

Outer Membrane Vesicles (OMV) have received increased attention in recent years as a vaccine platform against bacterial pathogens. OMV from Neisseria meningitidis serogroup B have been extensively explored. Following the success of the MeNZB OMV vaccine in controlling an outbreak of N. meningitidis B in New Zealand, additional research and development resulted in the licensure of the OMV-containing four-component 4CMenB vaccine, Bexsero. This provided broader protection against multiple meningococcal B strains. Advances in the field of genetic engineering have permitted further improvements in the platform resulting in increased yields, reduced endotoxicity and decoration with homologous and heterologous antigens to enhance immuno genicity and provide broader protection. The OMV vaccine platform has been extended to many other pathogens. In this review, we discuss progress in the development of the OMV vaccine delivery platform, highlighting successful applications, together with potential challenges and gaps.

Short Vi-polysaccharide abrogates T-independent immune response and hyporesponsiveness elicited by long Vi-CRM197 conjugate vaccine.

Polysaccharide-protein conjugates have been developed to overcome the T-independent response, hyporesponsiveness to repeated vaccination, and poor immunogenicity in infants of polysaccharides. To address the impact of polysaccharide length, typhoid conjugates made with short- and long-chain fractions of Vi polysaccharide with average sizes of 9.5, 22.8, 42.7, 82.0, and 165 kDa were compared. Long-chain-conjugated Vi (165 kDa) induced a response in both wild-type and T cell-deficient mice, suggesting that it maintains a T-independent response. In marked contrast, short-chain Vi (9.5 to 42.7 kDa) conjugates induced a response in wild-type mice but not in T cell-deficient mice, suggesting that the response is dependent on T cell help. Mechanistically, this was explained in neonatal mice, in which long-chain, but not short-chain, Vi conjugate induced late apoptosis of Vi-specific B cells in spleen and early depletion of Vi-specific B cells in bone marrow, resulting in hyporesponsiveness and lack of long-term persistence of Vi-specific IgG in serum and IgG+ antibody-secreting cells in bone marrow. We conclude that while conjugation of long-chain Vi generates T-dependent antigens, the conjugates also retain T-independent properties, leading to detrimental effects on immune responses. The data reported here may explain some inconsistencies observed in clinical trials and help guide the design of effective conjugate vaccines.

The Background, Role and Approach for Development of a Controlled Human Infection Model for Nontyphoidal Salmonella.

Nontyphoidal Salmonella (NTS) is responsible for a major global burden of disease and economic loss, particularly in low- and middle-income countries. It is designated a priority pathogen by the WHO for vaccine development and, with new impetus from vaccine developers, the establishment of an NTS controlled human infection model (CHIM) is timely and valuable. The broadly dichotomous clinical presentations of diarrhoea and invasive disease, commonly bacteraemia, present significant challenges to the development of an NTS CHIM. Nevertheless, if successful, such a CHIM will be invaluable for understanding the pathogenesis of NTS disease, identifying correlates of protection and advancing candidate vaccines towards licensure. This article describes the background case for a CHIM for NTS, the role of such a CHIM and outlines a potential approach to its development.

Impact and Control of Sugar Size in Glycoconjugate Vaccines.

Glycoconjugate vaccines have contributed enormously to reducing and controlling encapsulated bacterial infections for over thirty years. Glycoconjugate vaccines are based on a carbohydrate antigen that is covalently linked to a carrier protein; this is necessary to cause T cell responses for optimal immunogenicity, and to protect young children. Many interdependent parameters affect the immunogenicity of glycoconjugate vaccines, including the size of the saccharide antigen. Here, we examine and discuss the impact of glycan chain length on the efficacy of glycoconjugate vaccines and report the methods employed to size polysaccharide antigens, while highlighting the underlying reaction mechanisms. A better understanding of the impact of key parameters on the immunogenicity of glycoconjugates is critical to developing a new generation of highly effective vaccines.

Typhoid in India: An Age-old Problem With an Existing Solution.

Enteric fever continues to impact millions of people who lack adequate access to clean water and sanitation. The typhoid and paratyphoid fever burden in South Asia is broadly acknowledged, but current estimates of incidence, severity, and cost of illness from India are lacking. This supplement addresses this gap in our knowledge, presenting findings from two years of surveillance, conducted at multiple sites between October 2017 and February 2020, in the Surveillance for Enteric Fever in India (SEFI) network. Results provide contemporaneous evidence of high disease burden and cost of illness-the latter borne largely by patients in the absence of universal healthcare coverage in India. Against a backdrop of immediate priorities in the COVID-19 pandemic, these data are a reminder that typhoid, though often forgotten, remains a public health problem in India. Typhoid conjugate vaccines, produced by multiple Indian manufacturers, and recommended for use in high burden settings, ensure that the tools to tackle typhoid are an immediately available solution to this public health problem.

Comparative immunogenicity and efficacy of equivalent outer membrane vesicle and glycoconjugate vaccines against nontyphoidal Salmonella.

Nontyphoidal Salmonellae cause a devastating burden of invasive disease in sub-Saharan Africa with high levels of antimicrobial resistance. Vaccination has potential for a major global health impact, but no licensed vaccine is available. The lack of commercial incentive makes simple, affordable technologies the preferred route for vaccine development. Here we compare equivalent Generalized Modules for Membrane Antigens (GMMA) outer membrane vesicles and O-antigen-CRM197 glycoconjugates to deliver lipopolysaccharide O-antigen in bivalent Salmonella Typhimurium and Enteritidis vaccines. Salmonella strains were chosen and tolR deleted to induce GMMA production. O-antigens were extracted from wild-type bacteria and conjugated to CRM197 Purified GMMA and glycoconjugates were characterized and tested in mice for immunogenicity and ability to reduce Salmonella infection. GMMA and glycoconjugate O-antigen had similar structural characteristics, O-acetylation, and glucosylation levels. Immunization with GMMA induced higher anti-O-antigen IgG than glycoconjugate administered without Alhydrogel adjuvant. With Alhydrogel, antibody levels were similar. GMMA induced a diverse antibody isotype profile with greater serum bactericidal activity than glycoconjugate, which induced almost exclusively IgG1. Immunization reduced bacterial colonization of mice subsequently infected with SalmonellaS Typhimurium numbers were lower in tissues of mice vaccinated with GMMA compared with glycoconjugate. S. Enteritidis burden in the tissues was similar in mice immunized with either vaccine. With favorable immunogenicity, low cost, and ability to induce functional antibodies and reduce bacterial burden, GMMA offer a promising strategy for the development of a nontyphoidal Salmonella vaccine compared with established glycoconjugates. GMMA technology is potentially attractive for development of vaccines against other bacteria of global health significance.

Typhoid Conjugate Vaccines and Enteric Fever Control: Where to Next?

After the unprecedented success and acceleration of the global agenda towards typhoid fever control with a strong World Health Organization recommendation and the approval of funding from Gavi, the Vaccine Alliance (Gavi), for the use of a new typhoid conjugate vaccine (TCV), we should turn our minds to the challenges that remain ahead. Despite the evidence showing the safety and clinical efficacy of TCV in endemic populations in developing countries, we should remain vigilant and explore hurdles for the full public health impact of TCV, including vaccine supply for the potential global demand, immunization strategies to optimize the effectiveness and long-term protection provided by the vaccines, potential use of TCV in outbreak settings, and scenarios for addressing chronic carriers. Finally, challenges face endemic countries with poor surveillance systems concerning awareness of the need for TCV and the extent of the issue across their populations, and how to target immunization strategies appropriately.

Consensus Report on Shigella Controlled Human Infection Model: Introduction and Overview.

In recent years, controlled human infection models (CHIMs) have become available for a range of infectious agents and have proved invaluable for understanding the disease process, pathogenesis, and mechanisms of immunity. CHIM studies have also contributed significantly to advancing development of a number of vaccines by providing an indication of vaccine efficacy. The Shigella CHIM has been established in 3 sites in the United States, and it is likely that the CHIM will play an important regulatory role for advancing the range of Shigella vaccine candidates that are currently in development. This supplement describes the harmonization of best practices across sites, with a view to maximizing the contribution that CHIM studies can make to Shigella vaccine development.

Consensus Report on Shigella Controlled Human Infection Model: Clinical Endpoints.

The Shigella controlled human infection model (CHIM) is valuable for assessing candidate Shigella vaccine efficacy and potentially accelerating regulatory approval. The Shigella CHIM is currently being conducted at 3 sites in the United States using Shigella flexneri 2a strain 2457T and Shigella sonnei strain 53G. Shigellosis can present variably as watery diarrhea alone or with dysentery, and can be accompanied by manifestations including fever, abdominal cramps, tenesmus, and malaise. For comparability, it is important to harmonize the primary clinical endpoint. An expert working group was convened on 2 February 2018 to review clinical data from Shigella CHIM studies performed to date and to develop a consensus primary endpoint. The consensus endpoint enabled "shigellosis" to present as severe diarrhea or moderate diarrhea or dysentery. The latter 2 criteria are met when concurrent with fever of 38.0°C and/or vomiting, and/or a constitutional/enteric symptom graded at least as "moderate" severity. The use of a blinded independent committee to adjudicate the primary endpoint by subject was also regarded as important. As safety of volunteers in challenge studies is of paramount importance and treatment timing can affect primary outcomes, a standard for early antibiotic administration was established as follows: (1) when the primary endpoint is met; (2) if a fever of ≥39.0°C develops; or (3) if the study physician deems it appropriate. Otherwise, antibiotics are given at 120 hours postinfectious challenge. The working group agreed on objective and subjective symptoms to be solicited, and standardized methods for assessing subject-reported severity of symptoms.

Consensus Report on Shigella Controlled Human Infection Model: Immunological Assays.

Moderate to severe diarrhea caused by Shigella is a global health concern due to its substantial contribution to morbidity and mortality in children aged <5 years in low- and middle-income countries. Although antibiotic treatment can be effective, emerging antimicrobial resistance, limited access, and cost affirm the role of vaccines as the most attractive countermeasure. Controlled human infection models (CHIMs) represent a valuable tool for assessing vaccine efficacy and potentially accelerating licensure. Currently, immunological analysis during CHIM studies is customized based on vaccine type, regimen, and administration route. Additionally, differences in type of immunoassays and procedures used limit comparisons across studies. In November 2017, an expert working group reviewed Shigella CHIM studies performed to date and developed consensus guidelines on prioritization of immunoassays, specimens, and collection time points. Immunoassays were ranked into 3 tiers, with antibodies to Shigella lipopolysaccharide (LPS) being the highest priority. To facilitate comparisons across clinical studies, a second workshop was conducted in December 2017, which focused on the pathway toward a recognized enzyme-linked immunosorbent assay (ELISA) to determine serum immunoglobulin G titers against Shigella LPS. The consensus of the meeting was to establish a consortium of international institutions with expertise in Shigella immunology that would work with the National Institute for Biological Standards and Control to establish a harmonized ELISA, produce a reference sera, and identify a reliable source of Shigella LPS for global utilization. Herein we describe efforts toward establishing common procedures to advance Shigella vaccine development, support licensure, and ultimately facilitate vaccine deployment and uptake.

Consensus Report on Shigella Controlled Human Infection Model: Conduct of Studies.

Shigella causes morbidity and mortality worldwide, primarily affecting young children living in low-resource settings. It is also of great concern due to increasing antibiotic resistance, and is a priority organism for the World Health Organization. A Shigella vaccine would decrease the morbidity and mortality associated with shigellosis, improve child health, and decrease the need for antibiotics. Controlled human infection models (CHIMs) are useful tools in vaccine evaluation for early up- or down-selection of vaccine candidates and potentially useful in support of licensure. Over time, the methods employed in these models have become more uniform across sites performing CHIM trials, although some differences in conduct persist. In November 2017, a Shigella CHIM workshop was convened in Washington, District of Columbia. Investigators met to discuss multiple aspects of these studies, including study procedures, clinical and immunological endpoints, and shared experiences. This article serves as a uniform procedure by which to conduct Shigella CHIM studies.

The Severe Typhoid Fever in Africa Program: Study Design and Methodology to Assess Disease Severity, Host Immunity, and Carriage Associated With Invasive Salmonellosis.

BACKGROUND: Invasive salmonellosis is a common community-acquired bacteremia in persons residing in sub-Saharan Africa. However, there is a paucity of data on severe typhoid fever and its associated acute and chronic host immune response and carriage. The Severe Typhoid Fever in Africa (SETA) program, a multicountry surveillance study, aimed to address these research gaps and contribute to the control and prevention of invasive salmonellosis. METHODS: A prospective healthcare facility-based surveillance with active screening of enteric fever and clinically suspected severe typhoid fever with complications was performed using a standardized protocol across the study sites in Burkina Faso, the Democratic Republic of Congo (DRC), Ethiopia, Ghana, Madagascar, and Nigeria. Defined inclusion criteria were used for screening of eligible patients for enrollment into the study. Enrolled patients with confirmed invasive salmonellosis by blood culture or patients with clinically suspected severe typhoid fever with perforation were eligible for clinical follow-up. Asymptomatic neighborhood controls and immediate household contacts of each case were enrolled as a comparison group to assess the level of Salmonella-specific antibodies and shedding patterns. Healthcare utilization surveys were performed to permit adjustment of incidence estimations. Postmortem questionnaires were conducted in medically underserved areas to assess death attributed to invasive Salmonella infections in selected sites. RESULTS: Research data generated through SETA aimed to address scientific knowledge gaps concerning the severe typhoid fever and mortality, long-term host immune responses, and bacterial shedding and carriage associated with natural infection by invasive salmonellae. CONCLUSIONS: SETA supports public health policy on typhoid immunization strategy in Africa.

The essential role of complement in antibody-mediated resistance to Salmonella.

Vaccines can serve as essential tools to prevent bacterial diseases via the induction of long-lasting IgG responses. The efficacy of such vaccines depends on the effector mechanisms triggered by IgG. The complement system and Fc-gamma receptors (FcγRs) can potentially play a crucial role in IgG-mediated immunity against bacterial diseases. However, their relative importance in vivo is unclear, and has been the object of controversy and debate. In this brief study, we have used gene-targeted mice lacking either FcγRI, II, II and IV or the C3 complement component as well as a novel mouse strain lacking both C3 and FcγRs to conclusively show the essential role of complement in antibody-mediated host resistance to Salmonella enterica systemic infection. By comparing the effect of IgG2a antibodies against Salmonella O-antigen in gene-targeted mice, we demonstrate that the complement system is essential for the IgG-mediated reduction of bacterial numbers in the tissues.

IgG1 Is Required for Optimal Protection after Immunization with the Purified Porin OmpD from Salmonella Typhimurium.

In mice, the IgG subclass induced after Ag encounter can reflect the nature of the Ag. Th2 Ags such as alum-precipitated proteins and helminths induce IgG1, whereas Th1 Ags, such as Salmonella Typhimurium, predominantly induce IgG2a. The contribution of different IgG isotypes to protection against bacteria such as S. Typhimurium is unclear, although as IgG2a is induced by natural infection, it is assumed this isotype is important. Previously, we have shown that purified S. Typhimurium porins including outer membrane protein OmpD, which induce both IgG1 and IgG2a in mice, provide protection to S. Typhimurium infection via Ab. In this study we report the unexpected finding that mice lacking IgG1, but not IgG2a, are substantially less protected after porin immunization than wild-type controls. IgG1-deficient mice produce more porin-specific IgG2a, resulting in total IgG levels that are similar to wild-type mice. The decreased protection in IgG1-deficient mice correlates with less efficient bacterial opsonization and uptake by macrophages, and this reflects the low binding of outer membrane protein OmpD-specific IgG2a to the bacterial surface. Thus, the Th2-associated isotype IgG1 can play a role in protection against Th1-associated organisms such as S. Typhimurium. Therefore, individual IgG subclasses to a single Ag can provide different levels of protection and the IgG isotype induced may need to be a consideration when designing vaccines and immunization strategies.

Role of sapA and yfgA in Susceptibility to Antibody-Mediated Complement-Dependent Killing and Virulence of Salmonella enterica Serovar Typhimurium.

The ST313 pathovar of Salmonella enterica serovar Typhimurium contributes to a high burden of invasive disease among African infants and HIV-infected adults. It is characterized by genome degradation (loss of coding capacity) and has increased resistance to antibody-dependent complement-mediated killing compared with enterocolitis-causing strains of S Typhimurium. Vaccination is an attractive disease-prevention strategy, and leading candidates focus on the induction of bactericidal antibodies. Antibody-resistant strains arising through further gene deletion could compromise such a strategy. Exposing a saturating transposon insertion mutant library of S Typhimurium to immune serum identified a repertoire of S Typhimurium genes that, when interrupted, result in increased resistance to serum killing. These genes included several involved in bacterial envelope biogenesis, protein translocation, and metabolism. We generated defined mutant derivatives using S Typhimurium SL1344 as the host. Based on their initial levels of enhanced resistance to killing, yfgA and sapA mutants were selected for further characterization. The S Typhimurium yfgA mutant lost the characteristic Salmonella rod-shaped appearance, exhibited increased sensitivity to osmotic and detergent stress, lacked very long lipopolysaccharide, was unable to invade enterocytes, and demonstrated decreased ability to infect mice. In contrast, the S Typhimurium sapA mutants had similar sensitivity to osmotic and detergent stress and lipopolysaccharide profile and an increased ability to infect enterocytes compared with the wild type, but it had no increased ability to cause in vivo infection. These findings indicate that increased resistance to antibody-dependent complement-mediated killing secondary to genetic deletion is not necessarily accompanied by increased virulence and suggest the presence of different mechanisms of antibody resistance.

An O antigen capsule modulates bacterial pathogenesis in Shigella sonnei.

Shigella is the leading cause for dysentery worldwide. Together with several virulence factors employed for invasion, the presence and length of the O antigen (OAg) of the lipopolysaccharide (LPS) plays a key role in pathogenesis. S. flexneri 2a has a bimodal OAg chain length distribution regulated in a growth-dependent manner, whereas S. sonnei LPS comprises a monomodal OAg. Here we reveal that S. sonnei, but not S. flexneri 2a, possesses a high molecular weight, immunogenic group 4 capsule, characterized by structural similarity to LPS OAg. We found that a galU mutant of S. sonnei, that is unable to produce a complete LPS with OAg attached, can still assemble OAg material on the cell surface, but a galU mutant of S. flexneri 2a cannot. High molecular weight material not linked to the LPS was purified from S. sonnei and confirmed by NMR to contain the specific sugars of the S. sonnei OAg. Deletion of genes homologous to the group 4 capsule synthesis cluster, previously described in Escherichia coli, abolished the generation of the high molecular weight OAg material. This OAg capsule strongly affects the virulence of S. sonnei. Uncapsulated knockout bacteria were highly invasive in vitro and strongly inflammatory in the rabbit intestine. But, the lack of capsule reduced the ability of S. sonnei to resist complement-mediated killing and to spread from the gut to peripheral organs. In contrast, overexpression of the capsule decreased invasiveness in vitro and inflammation in vivo compared to the wild type. In conclusion, the data indicate that in S. sonnei expression of the capsule modulates bacterial pathogenesis resulting in balanced capabilities to invade and persist in the host environment.

Vaccines for low-income countries.

Low-income countries typically lag behind industrialised nations, where the introduction of new vaccines is commonly tailored to the pressures of the commercial market. Happily in recent years this paradigm has started to change with the introduction of a univalent meningococcal A conjugate vaccine that is specifically targeted for the prevention of epidemic meningitis in Africa. The declaration of the 2010s as a New Decade of Vaccines, together with Millennium Development Goals 4 and 5, provide a strong mandate for a new approach to the development of vaccines for low-income countries, so that there has never been a more exciting time to work in this field. This review considers the opportunities and challenges of developing these new vaccines in the context of innovations in vaccinology, the need to induce protective immunity in the populations at risk and the requirement for strong partnership between the countries that will use these vaccines and different elements of the vaccine industry.

Vaccines against poverty.

With the 2010s declared the Decade of Vaccines, and Millennium Development Goals 4 and 5 focused on reducing diseases that are potentially vaccine preventable, now is an exciting time for vaccines against poverty, that is, vaccines against diseases that disproportionately affect low- and middle-income countries (LMICs). The Global Burden of Disease Study 2010 has helped better understand which vaccines are most needed. In 2012, US$1.3 billion was spent on research and development for new vaccines for neglected infectious diseases. However, the majority of this went to three diseases: HIV/AIDS, malaria, and tuberculosis, and not neglected diseases. Much of it went to basic research rather than development, with an ongoing decline in funding for product development partnerships. Further investment in vaccines against diarrheal diseases, hepatitis C, and group A Streptococcus could lead to a major health impact in LMICs, along with vaccines to prevent sepsis, particularly among mothers and neonates. The Advanced Market Commitment strategy of the Global Alliance for Vaccines and Immunisation (GAVI) Alliance is helping to implement vaccines against rotavirus and pneumococcus in LMICs, and the roll out of the MenAfriVac meningococcal A vaccine in the African Meningitis Belt represents a paradigm shift in vaccines against poverty: the development of a vaccine primarily targeted at LMICs. Global health vaccine institutes and increasing capacity of vaccine manufacturers in emerging economies are helping drive forward new vaccines for LMICs. Above all, partnership is needed between those developing and manufacturing LMIC vaccines and the scientists, health care professionals, and policy makers in LMICs where such vaccines will be implemented.

The Relationship Between Invasive Nontyphoidal Salmonella Disease, Other Bacterial Bloodstream Infections, and Malaria in Sub-Saharan Africa.

BACKGROUND: Country-specific studies in Africa have indicated that Plasmodium falciparum is associated with invasive nontyphoidal Salmonella (iNTS) disease. We conducted a multicenter study in 13 sites in Burkina Faso, Ethiopia, Ghana, Guinea-Bissau, Kenya, Madagascar, Senegal, South Africa, Sudan, and Tanzania to investigate the relationship between the occurrence of iNTS disease, other systemic bacterial infections, and malaria. METHODS: Febrile patients received a blood culture and a malaria test. Isolated bacteria underwent antimicrobial susceptibility testing, and the association between iNTS disease and malaria was assessed. RESULTS: A positive correlation between frequency proportions of malaria and iNTS was observed (P = .01; r = 0.70). Areas with higher burden of malaria exhibited higher odds of iNTS disease compared to other bacterial infections (odds ratio [OR], 4.89; 95% CI, 1.61-14.90; P = .005) than areas with lower malaria burden. Malaria parasite positivity was associated with iNTS disease (OR, 2.44; P = .031) and gram-positive bacteremias, particularly Staphylococcus aureus, exhibited a high proportion of coinfection with Plasmodium malaria. Salmonella Typhimurium and Salmonella Enteritidis were the predominant NTS serovars (53/73; 73%). Both moderate (OR, 6.05; P = .0001) and severe (OR, 14.62; P < .0001) anemia were associated with iNTS disease. CONCLUSIONS: A positive correlation between iNTS disease and malaria endemicity, and the association between Plasmodium parasite positivity and iNTS disease across sub-Saharan Africa, indicates the necessity to consider iNTS as a major cause of febrile illness in malaria-holoendemic areas. Prevention of iNTS disease through iNTS vaccines for areas of high malaria endemicity, targeting high-risk groups for Plasmodium parasitic infection, should be considered.