IgG subclass distribution of antibodies after vaccination of adults with pneumococcal conjugate vaccines

The serum IgG subclass response of adults to Streptococcus pneumoniae (Pnc) capsular polysaccharides (PS) 6B, 14 and 23F was measured for four Pnc vaccines: the 23-valent PS vaccine or PS-protein conjugates with diphtheria toxoid (PncD), tetanus protein (PncT) or CRM197 protein (PncCRM) carriers. A standardized enzyme-linked immunosorbent assay specific for IgG subclasses was employed. This assay uses pneumococcal reference serum, lot 89-SF, to which anti-Pnc PS IgG subclass concentrations have been assigned. Both IgG1 and IgG2 responses were more frequent and higher in the conjugate groups than in the PS group. IgG subclasses in subjects vaccinated with PS displayed similar IgG2 predominant distribution previously observed in both natural and vaccine-induced antibodies. Antibodies induced by PncT, however, had a significantly altered IgG2/IgG1 ratio (P < 0.05), with a higher proportion of IgG1.     

Development of Shigella conjugate vaccines targeting Shigella flexneri 2a and S. flexneri 3a using a simple platform-approach conjugation by squaric acid chemistry

There is a need for vaccines effective against shigella infection in young children in resource-limited areas. Protective immunity against shigella infection targets the O-specific polysaccharide (OSP) component of lipopolysaccharide. Inducing immune responses to polysaccharides in young children can be problematic, but high level and durable responses can be induced by presenting polysaccharides conjugated to carrier proteins. An effective shigella vaccine will need to be multivalent, targeting the most common global species and serotypes such as Shigella flexneri 2a, S. flexneri 3a, S. flexneri 6, and S. sonnei. Here we report the development of shigella conjugate vaccines (SCV) targeting S. flexneri 2a (SCV-Sf2a) and 3a (SCV-Sf3a) using squaric acid chemistry to result in single point sun-burst type display of OSP from carrier protein rTTHc, a 52 kDa recombinant protein fragment of the heavy chain of tetanus toxoid. We confirmed structure and demonstrated that these conjugates were recognized by serotype-specific monoclonal antibodies and convalescent sera of humans recovering from shigellosis in Bangladesh, suggesting correct immunological display of OSP. We vaccinated mice and found induction of serotype-specific OSP and LPS IgG responses, as well as rTTHc-specific IgG responses. Vaccination induced serotype-specific bactericidal antibody responses against S. flexneri, and vaccinated animals were protected against keratoconjunctivitis (Sereny test) and intraperitoneal challenge with virulent S. flexneri 2a and 3a, respectively. Our results support further development of this platform conjugation technology in the development of shigella conjugate vaccines for use in resource-limited settings.     

Children with otitis media mount a pneumococcal serotype specific serum IgG and IgA response comparable to healthy controls after pneumococcal conjugate vaccination

It has been suggested that otitis-prone children have an impaired antibody response. To investigate this in the context of pneumococcal vaccination, we used a multiplex bead-based assay to measure serum IgG and IgA levels against pneumococcal serotypes included in the 7-valent pneumococcal conjugate vaccine (PCV7; serotypes 4, 6B, 9V, 14, 18C, 19F and 23F) and 4 non-PCV7 serotypes (1, 5, 7F and 19A) in healthy (n=43) and otitis-prone children (n=75) before, 6 weeks after and 1 year after vaccination with one dose of PCV7. Pre-vaccination, otitis-prone children had significantly higher serum IgG levels against serotypes 4, 9V and 23F and against all non-PCV7 serotypes. One year following vaccination, there was no difference in IgG or IgA levels between healthy and otitis-prone children. The effect of the administration of one or two doses of PCV7 was investigated in otitis-prone children. After a second dose of PCV7, pneumococcal serotype specific IgG levels, but not IgA titres, were higher compared to the levels measured after the initial dose of PCV7. One year post PCV7 vaccination there was no difference in either IgG or IgA antibody levels to any of the PCV7 serotypes between children who received either one or two doses of PCV7. The finding that otitis-prone children do not have an impaired pneumococcal serotype-specific serum IgG or IgA response suggests that new pneumococcal conjugate vaccines may be immunogenic in otitis-prone children, however, further investigations are necessary to determine the clinical impact of such vaccines against the development of recurrent acute otitis media.     

福氏2a痢疾结合疫苗临床安全性观察

目的:评价福氏2a痢疾结合疫苗临床安全性。方法:按随机、对照、盲法的原则,以不含福氏2a痢疾结合物疫苗成份的磷酸缓冲盐水作为安慰剂对照,开展现场临床试验,比较试验疫苗组和对照组免后反应率。结果:接种福氏2a痢疾结合疫苗2针后全身反应平均发生率为1.11%,其中弱反应占62.5%,中反应占37.5%,无强反应发生。局部反应平均发生率为8.89%。各年龄段试验疫苗组全身反应和局部反应发生率和对照组相比无统计学差异。结论:福氏2a痢疾结合疫苗在2岁以上的人群中接种是安全的。     

Live Shigella flexneri 2a and Shigella sonnei I vaccine candidate strains with two attenuating markers. II. Preliminary results of vaccination of adult volunteers and children aged 2-17 years

Shigella flexneri 2a and Shigella sonnei I vaccine candidate strains were tested for tolerance in 14 adults as well as for tolerance and immunogenicity in children aged 2-17 years. For fresh culture material, the limit for side effects fading within 36 h such as meteorism, loose stools, tenesmus and slight temperature, and tolerated by volunteers was 3 x 10(9) colony-forming units (c.f.u.). Excretion of vaccine strain germs for 2-3 days was observed only at doses of 10(10) c.f.u. and greater (2-3 x 10(10)). Children tolerated up to the maximum dose of 1-3 x 10(9) c.f.u. of the lyophilized vaccine without side effects. No vaccine strain germs were found in faeces, antigen being detected for 4-5 days. The total dose of 1-3 x 10(9) c.f.u. being distributed among three oral inoculations at intervals of 1-2 days induced coproantibodies in 70% of the vaccinees during the first 3 months, and in 30-40% up to 6 months. In contrast, the single application of this total dose for primary immunization, or booster after 10 months, proved to be more efficient. Ninety per cent of the subjects had markedly elevated titres for not less than 6 months.     

Development and evaluation of a Shigella flexneri 2a and S. sonnei bivalent invasin complex (Invaplex) vaccine

Over 160 million cases of shigellosis occur annually worldwide, with the two most prevalent species being Shigella flexneri and S. sonnei. Protective immunity against Shigella infection is primarily directed at the lipopolysaccharide (LPS) of the homologous serotype, so it may be necessary to combine monovalent vaccines for multiple Shigella serotypes to construct a multivalent vaccine against predominant serotypes. Recently, we described a subcellular vaccine isolated from virulent S. flexneri, consisting of proteins (including the invasins IpaB and IpaC) and LPS, that protected mice and guinea pigs from homologous challenge. In the present study, a bivalent Invaplex vaccine consisting of S. flexneri 2a and S. sonnei Invaplex was used to intranasally immunize mice and guinea pigs to determine the bivalent vaccine's immunogenicity and protective capacity against challenge with either strain. Mice and guinea pigs immunized with the bivalent S. flexneri 2a/S. sonnei Invaplex vaccine produced serum IgA and IgG antibodies to S. flexneri LPS, S. sonnei LPS, the homologous Invaplex and the water extract antigens (invasins) as determined by ELISA. The immune responses in animals immunized with the bivalent vaccine were similar to responses in animals immunized with the monovalent Invaplex vaccines. Mice and guinea pigs immunized with the bivalent vaccine were protected from a lethal lung challenge (mice, P<0.001) or severe keratoconjunctivitis (guinea pigs, P< or = 0.002) after challenge with either S. flexneri 2a or S. sonnei. Animals immunized with monovalent Invaplex vaccines were protected (P<0.001) against the homologous agent at levels comparable to the bivalent vaccine. After challenge, immunized animals demonstrated boosts in antibody titers to LPS, water extract antigens and Invaplex. These studies indicate that the subcellular Invaplex vaccine will be readily adaptable to a multivalent vaccine approach for shigellosis.     

Studies in volunteers to evaluate candidate Shigella vaccines: further experience with a bivalent Salmonella typhi-Shigella sonnei vaccine and protection conferred by previous Shigella sonnei disease

A bivalent vaccine consisting of Salmonella typhi strain Ty21a containing the 120 MDa plasmid of Shigella sonnei and expressing both S. typhi and S. sonnei lipopolysaccharides (LPS) on its surface was previously shown to protect significantly against S. sonnei disease in experimental challenge studies. However, protective efficacy could not be reconfirmed in volunteers with five subsequent lots of vaccine. One vaccine lot which resembled the initial protective lots of vaccine in biochemical and serological tests, and by electron microscopy, was administered to 16 volunteers who ingested three doses of 10(9) organisms each. Antibody secreting cells (ASC) specific for S. sonnei LPS were detected in the blood of 100% of vaccines, but no protection of these vaccines was demonstrated during a S. sonnei challenge study. To assess the ability of the volunteer model to detect infection-derived immunity, six volunteers who had had clinical shigellosis due to S. sonnei two months earlier were rechallenged with wild-type S. sonnei, together with 12 controls. Prior infection provided 100% protection against febrile illness (p = 0.05) and diarrhea (p = 0.04), thereby validating the volunteer model for assessing Shigella vaccines.     

Live Shigella flexneri 2a and Shigella sonnei I vaccine candidate strains with two attenuating markers. I. Construction of vaccine candidate strains with retained invasiveness but reduced intracellular multiplication

Live Shigella flexneri 2a and Shigella sonnei Phase I vaccine candidate strains with two virulence-reducing markers were constructed through stepwise incorporation of weakly attenuating purine auxotrophy with subsequent rifampicin-resistance (RNA polymerase) mutation to yield optimal attenuation. These vaccine candidate strains showed an unaltered plasmid profile; did not cause keratoconjunctivitis in the Sereny test, while being excreted for a short but still marked period and providing partial protection from wild-strain infection; exhibited for guinea-pig conjunctival epithelia, HeLa cells and rat enterocytes a maintained invasiveness with reduced intracellular multiplication with little, if any, reversible cell damage; and produced, just as their ultrasonic lysates, no exudative reaction in the rabbit gut loop test.     

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.     

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.     

Comparative analysis of influenza A(H3N2) virus hemagglutinin specific IgG subclass and IgA responses in children and adults after influenza vaccination

Two different influenza vaccines are generally used in many countries; trivalent live attenuated influenza vaccine (LAIV3) and trivalent inactivated influenza vaccine (IIV3). Studies comparing the antibody response to IIV3 and LAIV3 commonly investigate the seroprotective response by hemagglutination-inhibition (HI) assay. However, there is limited data regarding comparative analysis of IgG subclass and IgA responses induced by LAIV3 and IIV3.Fifteen children <5 years received 2 doses of LAIV3 while 14 children aged 10–17 years received one dose. In addition, 15 adults were vaccinated with either intranasal LAIV3 or intramuscular IIV3. We analyzed the H3N2 humoral responses by HI assay and the hemagglutinin (HA) specific IgG1, IgG2, IgG3, IgG4 and IgA1 responses by ELISA. Furthermore, we investigated the avidity of induced IgG antibodies.Pre-existing seroprotective HI antibodies were present in adults (73%) previously vaccinated with IIV3. Vaccination resulted in a significant increase in HI titers in all groups, except LAIV3 vaccinated adults. Furthermore, a negative correlation between age and HI titers in LAIV3 vaccinated subjects was observed post-vaccination. LAIV3 in children and IIV3 in adults induced HA-specific IgG1, low IgG3 but no IgG2 or IgG4. Moreover, significant IgA1 responses were only induced in children. Interestingly, IIV3 and LAIV3 induced IgG antibodies with comparable and significantly augmented avidity post-vaccination in children and adults.Our results suggest that age and/or exposure history play a significant role in determining the antibody response.Clinical trial registry: ClinicalTrials.gov NCT01003288 and NCT01866540     

福氏2a志贺氏菌肠毒素基因分型

目的：对福氏２ａ志贺氏菌（Ｓｈｉｇｅｌｌａ ｆｌｅｘｎｅｒｉ２ａ,Ｆ２ａ）的志贺氏肠毒素１（Ｓｈｉｇｅｌｌａ ｅｎｔｅｒｏｔｏｘｉｎ１,ＳＥＴ１）和志架氏肠毒素２（Ｓｈｉｇｅｌｌａ ｅｎｔｅｒｏｔｏｘｉｎ２,ＳＥＴ２）进行基因分型,提高菌痢暴发流行时同源性分析的水平。方法：对一起暴发Ｆ２ａ菌痢在现场流行病学调查的基础上,用聚合酶链反应（ＰＣＲ）方法,检测４３株Ｆ２ａ菌的志贺氏肠毒素１基因（ｓｅｔ１     

Studies on vaccination against bacillary dysentery. 3. Effective oral immunization against Shigella flexneri 2a in a field trial

In May 1963 a live vaccine prepared from streptomycin-dependent Shigella flexneri 2a was administered to 355 soldiers stationed in an area of Yugoslavia in which bacillary dysentery was hyperendemic. Five oral doses were given every third day. 382 unvaccinated soldiers from the same unit served as controls. Both groups were closely followed up until 16 September.     

Breastfeeding enhances the antibody response to Hib and Pneumococcal serotype 6B and 14 after vaccination with conjugate vaccines

BACKGROUND: This study was performed in order to investigate the relationship between breastfeeding and the antibody response after vaccination with conjugate vaccines against Hib and pneumococcal diseases. METHODS: This was an open non-randomised multi-centre study enrolling 101 healthy Swedish infants. PncCRM was administered concomitantly with DTaP/IPV/Hib at 3, 5, and 12 months at separate site. Duration of breastfeeding was calculated for days of almost exclusive as well as of total (any form of) breastfeeding. RESULTS: At 13 months of age 6 out of 83 children did not reach 0.2mug/ml against serotype 6B, and five of these were breastfed less than 90 days (Fisher's Exact test, P=0.011). Four children did not reach 1mug/ml against Hib and all those were breastfed less than 90 days (Fisher's Exact test, P=0.008). One month after the second dose, at 6 months of age, children breastfed 90 days or more showed significantly higher GMC against serotype 14 (P=0.003). CONCLUSION: This study indicates that children exclusively breastfed 90 days or more might get a better serological protection against Hib, and the pneumococcal serotypes 6B and 14 after vaccination, compared to children less breastfed.     

Pneumococcal conjugate vaccination does not induce a persisting mucosal IgA response in children with recurrent acute otitis media

AIM: In a prospective controlled study in young children with a history of recurrent acute otitis media, we analyzed the salivary IgA and IgG antibody titers upon vaccination with a 7-valent pneumococcal conjugate vaccine (PCV) given once or twice, followed by a 23-valent polysaccharide booster vaccination. METHODS: Salivary IgA and IgG antibody concentrations to vaccine serotype 6B, 14, 18C and 19F were measured by enzyme immunoassay in 38 samples of children vaccinated with PCV and 45 control samples. In the PCV group, 12 samples were taken prior to vaccination, 12 samples 4 weeks after the polysaccharide booster (8 months after the first conjugate vaccination) and 14 samples 7 months after the last vaccination (14 months after the first conjugate vaccination). In the control group 15 children were sampled at each of these three time points. RESULTS: We observed an increase in salivary IgG antibody concentrations against serotype 6B, 14, and 18C 14 months after the primary vaccination in children vaccinated with PCV twice, although this was significant for serotype 14 only. There was no increase in salivary IgG antibody in children vaccinate with PCV once nor in control children. IgA antibody titers increased significantly after 8 and after 14 months in both the pneumococcal vaccine recipients and the controls. However, the observed increase in mean antibody titers was significantly higher in control children compared to the PCV group. CONCLUSION: We suggest that repeated pneumococcal conjugate vaccination is necessary to induce an increase in salivary IgG antibodies and effectuate clearance of S. pneumoniae from the nasopharyngeal mucosa of children with recurrent acute otitis media. We hypothesize that the increase in salivary IgA is caused by the local boosting of the mucosal immune response by carriage and recurrent infections, which occurs less often in the PCV group compared to the control children.     

IgG antibody subclass responses determined by immunoblot in infants' sera following vaccination with a meningococcal recombinant hexavalent PorA OMV vaccine

The introduction of meningococcal serogroup C conjugate vaccines into the UK immunisation schedule has led to the decline of serogroup C disease in those vaccinated but there is no imminent vaccine solution for serogroup B disease. The PorA outer membrane protein (OMP) is a potential serogroup B vaccine candidate and an outer membrane vesicle (OMV) vaccine containing six different PorA OMPs (each representing a different serosubtype) has been evaluated in phase II trials with encouraging results. Little is known about the IgG subclass response to the various antigens contained within this vaccine. These responses are important due to the different half-lives and complement fixing abilities of these antibodies. In this study, immunoblotting was undertaken with infants' sera following either three or four doses of vaccine, and OMVs from six isogenic meningococcal strains differing only in their PorA serosubtype. Following either three or four doses of the vaccine, IgG(3) and IgG(1) subclass antibodies were induced to all six of the isogenic strains, although sera collected after four doses of vaccine showed stronger antibody levels. IgG(3) was found in more sera than IgG(1). For both sets of sera, the two isogenic strains expressing P1.5,2 and P1.5(c),10 induced stronger IgG subclass antibody responses than the other four meningococcal strains. The recombinant hexavalent PorA OMV vaccine stimulates both IgG(1) and IgG(3) subclass antibodies, the subclasses that are most effective in activating the complement system.     

Isolation of salmonellas and Shigella sonnei from a laboratory bench.

An area of the laboratory bench on which slide agglutinations were performed in the diagnosis of salmonella and shigella infection was examined for these organisms. Impression plates and broth-moistened swabs were used for sampling. Both techniques gave satisfactory results, but the contact plates provided positive results a day earlier than the swabs. Suitable precautions to minimize contamination of the bench surface are discussed.     

Technological investigations with attenuated strains of Shigella for production of live vaccines. 1. Cultivation in a fermenter of an attenuated strain S. flexneri 2a with two markers

A method was developed for cultivation of the strain Shigella flexneri 2a 77 with two attenuated markers. It ensures the preservation of the initial properties of the strain. The regimen for control of dissolved oxygen level was optimized. It was established that yeast extract is a necessary component of the nutritive medium used. The role of the phase and quantity of the inoculum for growth of the bacterial population has been clarified. Fresh fermenter culture, applied in rats, shows a high immunogenic activity. After lyophilization, the cultures retain 50-70% of the immunogenic activity of the non-lyophilized cultures. The method makes preconditions for the production of a live attenuated dysentery vaccine for oral administration.     

Construction and evaluation of a double mutant of Shigella flexneri as a candidate for oral vaccination against shigellosis

Based on studies on the genetic and molecular basis of Shigella flexneri invasive properties, we have constructed and evaluated a double mutant of S. flexneri serotype 5 for utilization as a live attenuated oral vaccine against shigellosis. The first mutation, icsA, blocks intracellular spread of bacteria as well as cell-to-cell infection. It affects the capacity of the invasive pathogen to form large abscesses in epithelia. The second mutation, iuc, eliminates production of the siderophore aerobactin thus impairing growth of the bacterium within tissues. This double mutant, SC5700 appeared safe when administered intragastrically to macaque monkeys as three doses (5 x 10(10) c.f.u. each) at weekly intervals. Protection against a challenge by the wild type isolate (M90T) was observed 4 weeks after the last vaccine inoculation. Duration of carriage was considerably reduced as compared to the control group in which all animals had developed severe dysentery. Seroconversion against serotype 5 LPS as well as S. flexneri virulence associated polypeptides was also observed.