Vaccination with Shigella flexneri 2a conjugate induces type 2a and cross-reactive type 6 antibodies in humans but not in mice

Shigella flexneri (S. flexneri) 6 has emerged as an important cause of shigellosis. Our efficacy study of Shigella sonnei and S. flexneri 2a O-specific polysaccharide (O-SP) conjugates in 1–4 year-olds had too few S. flexneri 2a cases for efficacy evaluation but surprisingly showed protection of 3–4 year-olds, S. flexneri 2a-recipients, from S. flexneri 6 infection. To investigate this cross-protection antibodies to both Shigella types were investigated in all sera remaining from previous studies. Twenty to 30% of 3–44 year-old humans injected with S. flexneri 2a conjugate responded with ≥4-fold increases of IgG anti type 6, p < 0.00001. The specificity of these antibodies was shown by inhibition studies. S. flexneri 6 infection of 2 children induced besides S. flexneri 6, also S. flexneri 2a antibodies, at levels of S. flexneri 2a vaccinees. S. flexneri 2a antibodies induced by S. flexneri 6 conjugates could not be studied since no such conjugate was assessed in humans and mice responded almost exclusively to the O-SP of the injected conjugate, with no cross-reactive antibodies.Our results indicate induction of cross-reactive protective antibodies. The O-acetylated disaccharide shared by S. flexneri 6 and 2a O-SPs, is the likely basis for their cross-reactivity. S. flexneri 6 O-SP conjugates, alone and in combination with S. flexneri 2a, merit further investigation for broad S. flexneri protection.     

Evaluation of a conjugate vaccine platform against enterotoxigenic Escherichia coli (ETEC), Campylobacter jejuni and Shigella

Enterotoxigenic Escherichia coli (ETEC), Campylobacter jejuni (CJ), and Shigella sp. are major causes of bacterial diarrhea worldwide, but there are no licensed vaccines against any of these pathogens. Most current approaches to ETEC vaccines are based on recombinant proteins that are involved in virulence, particularly adhesins. In contrast, approaches to Shigella and CJ vaccines have included conjugate vaccines in which Shigella lipopolysaccharides (LPS) or CJ capsule polysaccharides are chemically conjugated to proteins. We have explored the feasibility of developing a multi-pathogen vaccine by using ETEC proteins as conjugating partners for CJ and Shigella polysaccharides. We synthesized three vaccines in which two CJ polysaccharides were conjugated to two recombinant ETEC adhesins based on CFA/I (CfaEB) and CS6 (CssBA), and LPS from Shigella flexneri was also conjugated to CfaEB. The vaccines were immunogenic in mice as monovalent, bivalent and trivalent formulations. Importantly, functional antibodies capable of inducing hemaglutination inhibition (HAI) of a CFA/I expressing ETEC strain were induced in all vaccines containing CfaEB. These data suggest that conjugate vaccines could be a platform for a multi-pathogen, multi-serotype vaccine against the three major causes of diarrheal disease worldwide.     

Scalable production and immunogenicity of a cholera conjugate vaccine

There is a need to develop cholera vaccines that are protective in young children under 5 years of age, which induce long-term immunity, and which can be incorporated into the Expanded Programme of Immunization (EPI) in cholera-endemic countries. The degree of protection afforded by currently available oral cholera vaccines (OCV) to young children is significantly lower than that induced by vaccination of older vaccine recipients. Immune responses that protect against cholera target the O-specific polysaccharide (OSP) of Vibrio cholerae, and young children have poor immunological responses to bacterial polysaccharides, which are T cell independent antigens. To overcome this, we have developed a cholera conjugate vaccine (CCV) containing the OSP of V. cholerae O1, the main cause of endemic and epidemic cholera. Here, we describe production of CCV through a scalable manufacturing process and preclinical evaluation of immunogenicity in the presence and absence of aluminum phosphate (alum) as an adjuvant. The vaccine displays V. cholerae O1 Inaba OSP in sun-burst display via single point attachment of core oligosaccharide to a recombinant tetanus toxoid heavy chain fragment (rTTHc). Two different pilot-scale production batches of non-GMP CCV were manufactured and characterized in terms of physico-chemical properties and immunogenicity. In preclinical testing, the vaccine induced OSP- and lipopolysaccharide (LPS)-specific IgG and IgM responses, vibriocidal responses, memory B cell responses, and protection in a V. cholerae O1 challenge model. The addition of alum to the administered vaccine increased OSP-specific immune responses. These results support evaluation of CCV in humans.     

Intradermal administration of the pneumococcal conjugate vaccine in mice results in lower antibody responses as compared to intramuscular administration

IntroductionSeveral studies have shown that intradermal vaccination leads to improved immune responses. In addition, lowering vaccine doses will reduce costs and therefore potentially increase coverage. To determine whether intradermal delivery enhances the antibody responses against the 13-valent pneumococcal conjugate vaccine (PCV13), we compared intradermally and intramuscularly vaccinated mice.MethodsMice were immunized with PCV13, either intradermally or intramuscularly and CFU-counts in the nasal tissue were determined three or seven days after intranasal colonization with a serotype 4 clinical strain. Antibody concentrations against all thirteen polysaccharides were measured in blood and mucosal samples using a fluorescent-bead-based multiplex immunoassay.ResultsAntibody levels in both serum and mucosal samples were higher in the intramuscularly vaccinated group as compared to the intradermally vaccinated group. No protection against S. pneumoniae intranasal colonization was observed for either vaccination route.ConclusionsIntradermal vaccination was inferior to intramuscular immunization in inducing serotype-specific antibodies.     

Studies on vaccination against bacillary dysentery. 6. Protection of children by oral immunization with streptomycin-dependent Shigella strains

A field trial of oral streptomycin-dependent mutant Shigella vaccines in five hyperendemic areas of Yugoslavia in 1969 confirmed the findings of earlier studies by demonstrating the effectiveness of these vaccines against dysentery. For the first time, a high degree of protection was demonstrated in children. The vaccines induced serotype-specific immunity against Shigella flexneri 1 and 2a and S. sonnei. Postvaccinal reactions were minor and consisted of vomiting or diarrhoea, or both, in a small proportion of children within several hours of the administration of the vaccine. These reactions, seen mainly after the first dose, were dose-dependent and could be decreased by reducing the number of live organisms. Reactions to subsequent doses were much fewer. Pretreatment with sodium bicarbonate was necessary. Under the conditions of this study, the vaccines proved to be stable with no evidence of reversion of the mutant strains to the virulent parent.     

Development of meningococcal polysaccharide conjugate vaccine that can elicit long-lasting and strong cellular immune response with hepatitis B core antigen virus-like particles as a novel carrier protein

Neisseria meningitidis caused meningitis is life-threatening acute infection with high fatality and high frequency of severe sequelae. Meningococcal capsular polysaccharides can be used to prevent meningococcal disease; while conjugating the polysaccharides to carrier protein was found necessary to improve the immunogenicity and induce memory responses in infants and young children. Nevertheless, repeated administration of glycoconjugate vaccines might lead to carrier-induced epitope suppression due to limited number of carrier proteins. Here in this study, full-length hepatitis B core antigen virus-like particles (HBc VLPs) was used as a novel potential carrier protein for conjugation of meningococcal group C polysaccharides (CPS) with heterobifunctional polyethylene glycol (PEG) of different length (2, 5 and 10?kDa) as linkers. The physiochemical properties of the CPS-PEG-HBc conjugate vaccines were fully characterized. The TEM, DLS, native agarose gel electrophoresis, and HPLC analyses all confirmed the successful conjugation. As compared to plain CPS and the physical mixture of CPS and HBc, the immunization with the conjugate vaccines can generate about 10 times increase in CPS specific IgG titers with a significant boosting effect. HBc conjugation induced a shift to a Th1 cellular immune type response, as assessed by the increased IgG2a subclass production. In addition, vaccination of the conjugate vaccines elicited much enhanced avidity functional antibody and long-lasting immunological memory. IgG titers elicited by CPS-P2k-HBc, CPS-P5k-HBc and CPS-P10k-HBc at week 18 maintained 38.1%, 17.9% and 33.3% of their peak values. All these results demonstrated that HBc VLPs can be used as potential carrier protein to develop polysaccharide conjugate vaccines effective in eliciting long-lasting and strong cellular immune response.     

Immunization of newborns with bacterial conjugate vaccines

Bacterial conjugate vaccines are based on the principle of coupling immunogenic bacterial capsular polysaccharides to a carrier protein to facilitate the induction of memory T-cell responses. Following the success of Haemophilus influenzae type b conjugate vaccines in the 1980s, conjugate vaccines for Streptococcus pneumoniae and Neisseria meningitidis infections were developed and proven to be effective in protecting children against invasive disease. In this review, the use of conjugate vaccines in human newborns is discussed. Neonatal Haemophilus influenzae type b and pneumococcal conjugate vaccination schedules have been trialed and proven to be safe, with the majority of studies demonstrating no evidence for the induction of immune tolerance. Whether their neonatal administration also results in an earlier induction of clinical protection in the first 2–3 critical months of life is still to be demonstrated.     

Quantitative analysis of IgG class and subclass and IgA serum response to Shigella sonnei and Shigella flexneri 2a polysaccharides following vaccination with Shigella conjugate vaccines.

It has been recently reported that a conjugate vaccine composed of the O-specific polysaccharide of S. sonnei bound to Pseudomonas aeruginosa recombinant exoprotein A (rEPA) conferred 74% protection against S. sonnei shigellosis. In the present study affinity purified Shigella antibodies were used as standards to quantify and characterize the serum antibody response to vaccination with Shigella sonnei or Shigella flexneri 2a polysaccharide conjugated to rEPA. The geometric mean concentrations of antibodies at the pre-vaccination stage were 3.8 microg/ml for IgG anti-S. sonnei LPS and 11.26 microg/ml for IgG anti-S. flexneri 2a LPS. Vaccination with S. sonnei-rEPA and S. flexneri 2a-rEPA induced the production of specific IgG antibodies to levels of 115.8 microg/ml and 126.5 microg/ml, respectively. The levels of specific antibodies above the pre-vaccination values persisted for at least 2 years. The IgG response to S. flexneri 2a-rEPA conjugate was almost entirely represented by the IgG2 subclass. The concentration of IgG1 anti-S. sonnei LPS was significantly higher than that of IgG2 14 days after vaccination with the homologous conjugate, but decreased to similar levels to those of IgG2 6, 12 and 24 months after immunization. Since the only difference between the S. sonnei and S. flexneri 2a conjugates lies in the different polysaccharides of the two Shigella serogroups (the protein rEPA, is identical in both cases), it follows that the different pattern of IgG subclass response is a result of the different structures of the two O-polysaccharides of S. sonnei and S. flexneri 2a.     

Safety and immunogenicity of an intranasal Shigella flexneri 2a Invaplex 50 vaccine

Background

Shigella flexneri 2a lipopolysaccharide 50 is a nasally delivered subunit vaccine consisting of a macromolecular complex composed of LPS, IpaB, IpaC and IpaD. The current study examined vaccine safety and immunogenicity across a dose range and the clinical performance of a new intranasal delivery device.

Methods

Volunteers (N = 36) were randomized to receive vaccine via the Dolphin™ (Valois of America, Congers, New York) intranasal spray device at one of three doses (240, 480, and 690 μg) on days 0, 14, and 28. Another group (N = 8) received the 240 μg dose via pipette. Vaccine safety was actively monitored and antigen-specific humoral and mucosal immune responses were determined.

Results

There were no serious adverse events and the majority of adverse events (98%) were mild. Antibody secreting cells (ASC), plasma, and mucosal immune responses to Shigella antigens were detected at all three dose levels with the 690 μg dose inducing the highest magnitude and frequency of responses. Vaccination with comparable doses of Invaplex 50 via the Dolphin™ resulted in higher plasma and ASC immune responses as compared to pipette delivery.

Conclusion

In this trial the S. flexneri 2a Invaplex 50 vaccine was safe, well-tolerated and induced robust levels of antigen-specific intestinal IgA and ASC responses. The spray device performed well and offered an advantage over pipette intranasal delivery.     

Comparative effects of carrier proteins on vaccine-induced immune response

The efficacy of vaccines against major encapsulated bacterial pathogens - Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenzae type b (Hib) - has been significantly enhanced by conjugating the respective polysaccharides with different carrier proteins: diphtheria toxoid; non-toxic cross-reactive material of diphtheria toxin₁₉₇, tetanus toxoid, N. meningitidis outer membrane protein, and non-typeable H. influenzae-derived protein D. Hib, meningococcal, and pneumococcal conjugate vaccines have shown good safety and immunogenicity profiles regardless of the carrier protein used, although data are conflicting as to which carrier protein is the most immunogenic. Coadministration of conjugate vaccines bearing the same carrier protein has the potential for inducing either positive or negative effects on vaccine immunogenicity (immune interference). Clinical studies on the coadministration of conjugate vaccines reveal conflicting data with respect to immune interference and vaccine efficacy.     

Quality of antibody responses by adults and young children to 13-valent pneumococcal conjugate vaccination and Streptococcus pneumoniae colonisation

Childhood pneumococcal conjugate vaccine (PCV) protects against invasive pneumococcal disease caused by vaccine-serotype (VT) Streptococcus pneumoniae by generating opsonophagocytic anti-capsular antibodies, but how vaccination protects against and reduces VT carriage is less well understood. Using serological samples from PCV-vaccinated Malawian individuals and a UK human challenge model, we explored whether antibody quality (IgG subclass, opsonophagocytic killing, and avidity) is associated with protection from carriage. Following experimental challenge of adults with S. pneumoniae serotype 6B, 3/21 PCV13-vaccinees were colonised with pneumococcus compared to 12/24 hepatitis A-vaccinated controls; PCV13-vaccination induced serotype-specific IgG, IgG1, and IgG2, and strong opsonophagocytic responses. However, there was no clear relationship between antibody quality and protection from carriage or carriage intensity after vaccination. Similarly, among PCV13-vaccinated Malawian infants there was no relationship between serotype-specific antibody titre or quality and carriage through exposure to circulating serotypes. Although opsonophagocytic responses were low in infants, antibody titre and avidity to circulating serotypes 19F and 6A were maintained or increased with age. These data suggest a complex relationship between antibody-mediated immunity and pneumococcal carriage, and that PCV13-driven antibody quality may mature with age and exposure.     

Oral immunization with LacVax® OmpA induces protective immune response against Shigella flexneri 2a ATCC 12022 in a murine model

Shigellosis is an acute invasive disease of the lower intestine, which afflicts millions of people worldwide with an estimated one million fatalities per annum. Despite of extensive research during the last two decades, a vaccine against multi-drug resistant Shigella is not yet available in the market. To provide a safe, effective and broad-spectrum vaccine against Shigella, we explored food grade bacteria Lactococcus lactis (L. lactis) for the delivery of conserved antigenic protein; Outer membrane protein A (OmpA) to the mucosal sites for effective elicitation of systemic and mucosal immunity. We have previously confirmed the immunogenic potential of recombinant L. lactis expressing OmpA (LacVax® OmpA) in BALB/c mice. In the present study, we have characterized the humoral and cellular immune profile of LacVax® OmpA and assessed its protective efficacy using a newly developed human like murine shigellosis model. The significant increase in OmpA specific serum IgG, fecal sIgA and a Th1 dominant immune response (indicated by high INF-γ/IL-4 ratio) in LacVax® OmpA immunized mice revealed successful activation of humoral and cellular immunity. The LacVax® OmpA immunized animals were also protected from human-like shigellosis when challenged with S. flexneri 2a ATCC 12022. The antigen specific serum IgG, fecal sIgA, INF-γ and IL-10 levels were found to be the significant correlates of protection. Collectively these results suggest that the LacVax® OmpA is a promising prophylactic candidate against shigellosis. However, the protective efficacy of LacVax® OmpA in the higher animals would further strengthen its future application in humans.     

Pre-clinical evaluation of a 15-valent pneumococcal conjugate vaccine (PCV15-CRM197) in an infant-rhesus monkey immunogenicity model

The incidence of invasive pneumococcal disease (IPD), caused by the approximately 91 serotypes of Streptococcus pneumoniae (PN), varies geographically and temporally as a result of changing epidemiology and vaccination patterns as well as due to regional measurement differences. Prevnar^® (Pfizer), the first licensed pneumococcal conjugate vaccine (PCV), comprises polysaccharides (PS) from 7 serotypes conjugated to the mutant diphtheria toxin carrier protein, CRM197. In the United States and elsewhere, this vaccine has been highly efficacious in reducing the incidence of IPD caused by vaccine serotypes, however, the incidence of non-vaccine serotypes (e.g., 19A, 22F, and 33F) has increased, resulting in the need for vaccines with higher valencies. In response, 10- and 13-valent PCVs have recently been licensed. To further increase serotype coverage, we have developed a 15-valent PCV containing PS from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F conjugated to CRM197 and formulated on aluminum phosphate adjuvant. Vaccine immunogenicity was evaluated in infant rhesus monkeys since they, like human infants, respond poorly to unconjugated PN PS. Infant (2-3 month old) rhesus monkeys were vaccinated three times with PCV-15 or Prevnar^® at 2 month intervals, and serotype-specific IgG antibodies were measured using a multiarray electrochemiluminescence (ECL) assay. The results indicate that antibody responses to PCV-15 and Prevnar^® were comparable for the 7 common serotypes and that post-vaccination responses to PCV-15 were >10-fold higher than baseline for the 8 additional serotypes.     

Impaired serotype-specific immune function following pneumococcal vaccination in infants with prior carriage

The impact of prior nasopharyngeal carriage on serotype-specific IgG responses following immunization with pneumococcal conjugate vaccines (PCV) has recently been described. This report extends these findings to describe the attenuation of functional immune responses following 23-valent pneumococcal polysaccharide vaccination (PPS). We report the attenuation of immune responses following booster with the 23-valent pneumococcal polysaccharide vaccination (PPS) in infants with prior nasopharyngeal carriage of Streptococcus pneumoniae. Fijian infants who were part of a phase II randomized, controlled trial of reduced dose PCV7 schedules were the basis of this study. Pneumococcal carriage was determined at 6, 9 and 12 months of age, prior to PPS immunization. Serum samples collected at 18 weeks (post-PCV7), 12 months (pre-PPS), 12.5 months and 17 months (post-PPS) of age were assessed for serotype-specific IgG and opsonophagocytic responses.     

A Contribution of MdfA to Resistance to Fluoroquinolones in Shigella flexneri

In this study, we measured the drug resistance conferred by mdfA mutations in two Shigella flexneri strains. A mutant in mdfA genes was constructed by polymerase chain reaction–based, one-step inactivation of chromosomal genes. The antimicrobial susceptibility of parent and mutant strains to fluoroquinolones was determined by minimal inhibitory concentration (MICs). The △mdfA mutants were somewhat more susceptible to fluoroquinolones than the parent strains. The low level changes in MICs of the △mdfA mutants suggest that mdfA contributed the fluoroquinolone resistance in S flexneri. This finding found that the increased expression level of an MdfA efflux pump mediated fluoroquinolone resistance, but it is not likely a major effecter of higher resistance levels.     

Comparative evaluation of the antibody in lymphocyte supernatant (ALS) and enzyme-linked immunospot (ELISPOT) assays for measuring mucosal immune responses to Shigella antigens

Accurately assessing mucosal immune responses to candidate vaccines remains a technical challenge. ELISPOT is widely used as a surrogate of mucosal immune response by directly enumerating circulating antibody secreting cells (ASCs), while antibody in lymphocyte supernatant (ALS) titers the total amount of antibody secreted by ASC ex vivo using ELISA. ALS is more practical than ELISPOT because the ASC supernatant is frozen for ELISA that can be conducted at any time, with any antigen, and in any laboratory. We compared IgA and IgG responses to serotype-specific Shigella LPS using ELISPOT and ALS in subjects following vaccination or infection with Shigella. ALS results correlated well with ELISPOT results, and the ALS method was both sensitive and specific for the detection of antibody responses against Shigella LPS. Based on these observations, the ALS assay is a practical and flexible alternative to ELISPOT for measuring mucosal IgA responses to Shigella LPS antigen.     

Serotype of Streptococcus pneumoniae capsular polysaccharide can modify the Th1/Th2 cytokine profile and IgG subclass response to pneumococal-CRM(197) conjugate vaccines in a murine model

The cellular and antibody responses to type 14 and type 19F Streptococcus pneumoniae capsular polysaccharides (PS) conjugated to CRM(197) were investigated in a mouse model developed for pre-clinical evaluation and quality control of pneumococcal conjugate vaccines. Total IgG antibody and IgG subclasses against PS and the carrier protein for both conjugates were measured in addition to the T cell proliferation and cytokine profiles induced by these conjugates. While unconjugated PS 14 and 19F were at best only weakly immunogenic, both types of conjugate induced strong primary and secondary IgG responses to PS. The responses induced by the two conjugates to the carrier protein were very different; a high level of anti-CRM(197) IgG was induced only by the PS19F conjugate whereas a very weak response was induced by the PS14 conjugate. Interestingly, the IgG subclass distribution was different for the two conjugates; for PS19F conjugate, the IgG response was almost completely of IgG1 subclass with low levels of IgG3 and IgG2a while the response to PS14 conjugate was mainly of the IgG1 and IgG2a subclasses with a low level of IgG3. The anti-CRM(197) IgG subclass distribution was identical with that to the corresponding conjugated PS. Both types of conjugate induced strong T cell proliferation to recall antigens but induced different patterns of cytokine response in immune spleen cells which were indicative of a Th0 response or a mixture of Th1 and Th2 responses with a bias towards Th2 response in PS19F-CRM(197) immunised mice. In conclusion, PS14- and PS19F-CRM(197) conjugates induced different IgG subclass patterns as a result of inducing different patterns of cytokine response to the carrier protein. This indicates that the serotype of PS can modify the Th1/Th2 response to the carrier protein, which has a direct effect and can predict the IgG subclass of the PS response. Finally, we conclude that this model appears suitable for studying the immunogenicity and immune interaction of different components of multivalent pneumococcal conjugate vaccines and may be applicable to their pre-clinical evaluation and quality control.     

Chronic helminth infections impair pneumococcal vaccine responses

Pneumonia is the leading killer of children and disproportionately affects developing countries. Vaccination campaigns against Streptococcus pneumoniae, the leading cause of pneumonia, have recently been launched with a new conjugate vaccine in Africa. Using a mouse model, we assessed the potential role that the high burden of helminth infections in the countries targeted for vaccine might have on vaccine effectiveness. Mice vaccinated with either commercial conjugate or purified polysaccharide vaccines had impaired antibody responses if they were chronically infected with Taenia crassiceps. This translated to increased susceptibility to pneumococcal pneumonia and high mortality compared to helminth-negative vaccinated animals, which were fully protected from disease and death. Antibodies taken from Taenia-infected, vaccinated mice were unable to effectively opsonize S. pneumoniae for killing by alveolar macrophages, and did not protect against pneumococcal challenge when adoptively transferred into naïve animals. These data may have implications for vaccination programs in countries endemic with helminths.