Potent tetravalent replicon vaccines against botulinum neurotoxins using DNA-based Semliki Forest virus replicon vectors

Human botulism is commonly associated with botulinum neurotoxin (BoNT) serotypes A, B, E and F. This suggests that the greatest need is for a tetravalent vaccine that provides protection against all four of these serotypes. In current study, we investigated the feasibility of generating several tetravalent vaccines that protected mice against the four serotypes. Firstly, monovalent replicon vaccine against BoNT induced better antibody response and protection than that of corresponding conventional DNA vaccine. Secondly, dual-expression DNA replicon pSCARSE/FHc or replicon particle VRP-E/FHc vaccine was well resistant to the challenge of BoNT/E and BoNT/F mixture as a combination vaccine composed of two monovalent replicon vaccines. Finally, the dual-expression DNA replicon or replicon particle tetravalent vaccine could simultaneously and effectively neutralize and protect the four BoNT serotypes. Protection correlated directly with serum ELISA titers and neutralization antibody levels to BoNTs. Therefore, replicon-based DNA or particle might be effective vector to develop BoNT vaccines, which might be more desirable for use in clinical application than the conventional DNA vaccines. Our studies demonstrate the utility of combining dual-expression DNA replicon or replicon particle vaccines into multi-agent formulations as potent tetravalent vaccines for eliciting protective responses to four serotypes of BoNTs.     

Vaccination of cattle with a recombinant bivalent toxoid against botulism serotypes C and D

Cattle botulism is a fatal intoxication caused by botulinum neurotoxins (BoNTs) produced by Clostridium botulinum serotypes C and D resulting in economic losses. Vaccination is the most effective way to control botulism. However, the commercially available vaccines are difficult and hazardous to produce. Neutralizing antibodies against the C-terminal fragment of the BoNT heavy chain (H_C) are known to protect against lethal doses of BoNTs. We report the vaccination of cattle with a previously tested recombinant chimera consisting of Escherichia coli heat-labile enterotoxin B subunit and the H_C of BoNTs C and D. Vaccinated animals produced neutralizing antibodies against serotypes C and D averaging 5 ± 0 and 6.14 ± 1.06 IU/mL, respectively. For BoNT D, the titers were greater than those measured for the commercial vaccine, which induced titers of 5 ± 0 and 2.85 ± 1.35 against the respective serotypes, suggesting that this chimera is effective against cattle botulism.     

Total and serotype-specific pneumococcal antibody titres in children with normal and abnormal humoral immunity

A heptavalent pneumococcal conjugate vaccine (PCV-7) protects children against invasive pneumococcal disease. The aim of this study was to evaluate immunoglobulin subclass and serotype-specific pneumococcal antibody responses to vaccination in children with a history of recurrent or severe bacterial infections. Pneumococcal IgG, IgG1, IgG2 titres were assayed by ELISA, and nine serotype concentrations measured using a nonaplex bead assay in 145 children investigated for recurrent or severe infections. Children mounted an exclusively IgG1 response after vaccination with two doses of PCV-7 and a dose of 23 valent pneumococcal polysaccharide vaccine (PPV-23), with pneumococcal IgG2 antibody titres remaining low to negligible. Measurement of serotype-specific responses demonstrated that although PCV-7 specific serotype responses increased significantly post-vaccination, specific IgG against two of the serotypes not covered by PCV-7 but only by PPV-23 remained low. We conclude that in contrast to antibody response to natural infection with Pneumococcus or pneumococcal polysaccharide vaccines which are often of a IgG2 subclass, responses in children after PCV-7 are of IgG1 subclass. Serotype-specific IgG were useful in determining the protection against specific pneumococcal strains, and showed that the PPV-23 did not broaden protection against non-PCV-7 serotypes.     

Enhancing toxin-based vaccines against botulism

Botulinum neurotoxins (BoNT) are the most toxic proteins for humans. BoNTs are single chain proteins with an N-terminal light chain (LC) and a C-terminal heavy chain (HC). HC comprises a translocation domain (HC_N) and a receptor binding domain (HC_C). Currently, there are no approved vaccines against botulism. This study tests a recombinant, full-length BoNT/A1 versus LCHC_N/A1 and HC_C/A1 as vaccine candidates against botulism. Recombinant, full-length BoNT/A1 was detoxified by engineering 3-amino acid mutations (E224A/R363A/Y366F) (M-BoNT/A1) into the LC to eliminate catalytic activity, which reduced toxicity in a mouse model of botulism by >10⁶-fold relative to native BoNT/A1. As a second step to improve vaccine safety, an additional mutation (W1266A) was engineered in the ganglioside binding pocket, resulting in reduced receptor binding, to produce M-BoNT/A1^W. M-BoNT/A1^W vaccination protected against challenge by 10⁶ LD₅₀ Units of native BoNT/A1, while M-BoNT/A1 or M-BoNT/A1^W vaccination equally protected against challenge by native BoNT/A2, a BoNT subtype. Mice vaccinated with M-BoNT/A1^W surviving BoNT challenge had dominant antibody responses to the LCHC_N domain, but varied antibody responses to HC_C. Sera from mice vaccinated with M-BoNT/A1^W also neutralized BoNT/A1 action on cultured neuronal cells. The cell- and mouse-based assays measured different BoNT-neutralizing antibodies, where M-BoNT/A1^W elicited a strong neutralizing response in both assays. Overall, M-BoNT/A1^W, with defects in multiple toxin functions, elicits a potent immune response to BoNT/A challenge as a vaccine strategy against botulism and other toxin-mediated diseases.     

Head-to-head comparison of four nonadjuvanted inactivated cell culture-derived influenza vaccines: effect of composition, spatial organization and immunization route on the immunogenicity in a murine challenge model

In order to study the influence of antigen composition, spatial organization of antigen and the route of administration, four cell culture-derived, inactivated, nonadjuvanted influenza vaccine formulations, i.e. whole inactivated virus (WIV), split, subunit and virosome vaccines were prepared from a single antigen batch. We directly compared the immunogenicity and efficacy of these vaccine formulations after intramuscular (i.m.) or intranasal (i.n.) administration in mice. Prime and boost vaccination were followed by a potentially lethal homologous aerosol challenge. For all vaccines, the i.m. route induced higher serum humoral immune responses as compared to the i.n. route and protected all mice against challenge at a dose of 5 microg. Upon i.n. immunization only WIV and split vaccines induced detectable IgG titers and partial protection against challenge but only very low HI titers were induced in almost all mice. WIV induced mainly IgG2a/c titers via both routes, whereas split vaccine induced exclusively IgG1 titers via both routes. Subunit and virosome vaccines induced exclusively IgG1 via the i.m. route. Mucosal sIgA levels were only detected after i.n. vaccination with WIV. Furthermore, vaccines containing all viral components (WIV and split vaccine) induced higher serum HI titers and serum antibody titers than subunit and virosome vaccines. The differences in magnitude and quality of immune responses of split and WIV, having the same composition, are likely related to their distinct spatial organization. In conclusion, the direct comparison between WIV, split, subunit and virosomes, shows that the differences in immune responses between these well known influenza vaccines can be explained by both the composition and particulate structure of these vaccine formulations.     

Avidity and subtyping of specific antibodies applied to the indirect assessment of heterologous protection against Foot-and-Mouth Disease Virus in cattle

Serological assessment of the heterologous response among Foot-and-Mouth Disease Virus (FMDV) strains is mainly performed by virus neutralization test (VNT), liquid phase blocking ELISA and complement fixation assay. In this study two high-throughput ELISA techniques, avidity and IgG subtype ELISA, were developed and used to further characterize heterologous antibody responses in cattle during vaccination and challenge. Both assays were applied to a set of previously characterized sera from animals immunized with an inactivated A24 Cruzeiro/Brazil/55 (A24 Cruzeiro) strain monovalent FMDV vaccine and challenged with the heterologous A/Argentina/2001 (A/Arg/01) strain. Single dilution avidity ELISA assessment showed that animals that were protected against A/Arg/01 challenge had higher avidity antibodies to this heterologous strain than non-protected cattle. Animals with low or even undetectable anti-A/Arg/01 serum-neutralizing titers that passed the heterologous challenge presented higher IgG1/IgG2 ratio than non-protected animals. In this study, the three assessments (VNT and both ELISAs) discriminated between protected and not protected animals against a heterologous challenge. The combination of these techniques may be applied to complement current indirect serological vaccine-matching assessments. The measurement of these qualitative parameters may provide additional information to understand the mechanisms underlying FMD heterologous responses and the induction of cross-protection in cattle.     

Quantitative comparison of the cross-protection induced by tick-borne encephalitis virus vaccines based on European and Far Eastern virus subtypes

Tick-borne encephalitis virus (TBEV) is a flavivirus of wide geographic distribution and the causative agent of tick-borne encephalitis (TBE), an infection of the central nervous system. TBE has the highest incidence rate in Russia, where locally produced as well as Western European vaccines for the prevention of TBE are available. The Western European vaccines are based on TBE viruses that belong to the European subtype, while the Russian vaccines are based on Far Eastern subtype viruses. The question of to which extent vaccination with a vaccine based on the European subtype is effective in protecting against the heterologous Far Eastern virus subtype - and vice versa - has not been answered conclusively. Here we immunized mice with TBE vaccines based on European and Far Eastern subtype viruses, and used an unbiased hybrid virus test system to determine cross-neutralizing antibody titers and cross-protective efficacy. All vaccines tested elicited cross-protective responses against the heterologous strains, similar to those induced against the respective homologous vaccine strains. These data, therefore, fully support the use of TBE vaccines in geographic regions where virus subtypes heterologous to the vaccine strains are prevalent.     

Botulinum neurotoxin neutralizing activity of immune globulin (IG) purified from clinical volunteers vaccinated with recombinant botulinum vaccine (rBV A/B)

The basis for efficacy of the recombinant botulinum vaccine, serotypes A and B (rBV A/B) is that neutralizing antibodies induced by vaccination bind to botulinum neurotoxin complex serotype A, subtype A1 (BoNT/A1) and serotype B, subtype B1 (BoNT/B1) and prevent their actions at cholinergic neurons. The protective capacity of BoNT/A1 and BoNT/B1 neutralizing antibodies derived from the serum of clinical volunteers vaccinated with rBV A/B was evaluated in a guinea pig passive transfer model and a mouse bioassay. Guinea pigs passively immunized to achieve circulating neutralizing antibody concentration (NAC) levels representing the lowest measurable concentrations for BoNT/A1 and BoNT/B1 were protected against an intramuscular (IM) challenge more than 10 times the guinea pig IM median lethal dosage for BoNT/A1 and BoNT/B1. The passively immunized guinea pigs were asymptomatic during the 14-day post-challenge observation period. Control guinea pigs died within 48 h after challenge. Calculation of neutralizing efficiency of antibodies using results from a mouse bioassay indicated that a simple linearly proportional relationship does not exist between NAC level and the amount of BoNT neutralized. Based on this finding, estimates of level of protection must consider variability in BoNT neutralizing efficiency at different NAC levels. The protective capacity of human BoNT/A1 and BoNT/B1 neutralizing antibodies induced by rBV A/B vaccination was verified in a guinea pig passive immunization model. Additionally, estimates of the neutralizing efficiency have been established for BoNT/A1 and BoNT/B1 neutralizing antibodies obtained from clinical volunteers vaccinated with the rBV A/B.     

Protective efficacy of recombinant bacterin vaccine against botulism in cattle

Botulism is a paralytic disease caused by the intoxication of neurotoxins produced by Clostridium botulinum. Among the seven immunologically distinct serotypes of neurotoxins (BoNTs A − G), serotypes C and D, or a chimeric fusion termed C/D or D/C, are responsible for animal botulism. The most effective way to prevent botulism in cattle is through vaccination; however, the commercially available vaccines produced by detoxification of native neurotoxins are time-consuming and hazardous. To overcome these drawbacks, a non-toxic recombinant vaccine was developed as an alternative. In this study, the recombinant protein vaccine was produced using an Escherichia coli cell-based system. The formaldehyde-inactivated E. coli is able to induce 7.45 ± 1.77 and 6.6 ± 1.28 IU/mL neutralizing mean titers against BoNTs C and D in cattle, respectively, determined by mouse neutralization bioassay, and was deemed protective by the Brazilian legislation. Moreover, when the levels of anti-BoNT/C and D were compared with those achieved by the recombinant purified vaccines, no significant statistical difference was observed. Cattle vaccinated with the commercial vaccine developed 1.33 and 3.33 IU/mL neutralizing mean titers against BoNT serotypes C and D, respectively. To the best of our knowledge, this study is the first report on recombinant E. coli bacterin vaccine against botulism. The vaccine was safe and effective in generating protective antibodies and, thus, represents an industry-friendly alternative for the prevention of cattle botulism.     

Nanoemulsion W805EC improves immune responses upon intranasal delivery of an inactivated pandemic H1N1 influenza vaccine

Currently available influenza vaccines provide suboptimal protection. In order to improve the quality of protective immune responses elicited following vaccination, we developed an oil-in-water nanoemulsion (NE)-based adjuvant for an intranasally-delivered inactivated influenza vaccine. Using a prime-boost vaccination regimen, we show that intranasal vaccines containing the W₈₀5EC NE elicited higher titers of serum hemagglutination inhibiting (HAI) antibody and influenza-specific IgG and IgA titers compared to vaccines that did not contain the NE. Similarly, vaccines containing the W₈₀5EC NE resulted in higher influenza-specific IgA levels in the bronchoalveolar lavage (BAL) fluid and nasal wash when compared to vaccines formulated without NE. The higher antibody titers in mice immunized with the NE-containing vaccines correlated with reduced viral loads in the lungs and nasal turbinates following a high dose viral challenge. Mice immunized with vaccines containing the W₈₀5EC NE also showed a reduction in body weight loss following challenge compared to mice immunized with equivalent vaccines produced without NE. Taken together, our results show that the W₈₀5EC NE substantially improves the magnitude of protective influenza-specific antibody responses and is a promising mucosal adjuvant for influenza vaccines and vaccines against other mucosal pathogens.     

Bacterium-like particles supplemented with inactivated influenza antigen induce cross-protective influenza-specific antibody responses through intranasal administration

Administration of influenza vaccines through the intranasal (IN) route forms an attractive alternative to conventional intramuscular (IM) injection. It is not only a better accepted form of vaccine administration but it also has the potential to induce, in addition to systemic antibodies, local protective antibodies, i.e. S-IgA. Most commercially available vaccines however are inactivated non-replicating vaccines and have a low immunogenicity when administered intranasally. Local administration of these vaccines would therefore need an adjuvant to boost systemic and local antibody responses. Here we explored the use of a safe adjuvant system, i.e. bacterium-like particles (BLPs) derived from the food-grade bacterium in Lactococcus lactis, in the induction of protective antibody responses after intranasal immunization of mice. Supplementation of H1N1 split vaccine with BLPs significantly increased levels of serum influenza-specific IgG and hemagglutination-inhibiting antibodies: this was dependent on the dose of admixed BLPs and number of immunizations. Admixing BLPs further boosted local influenza-specific S-IgA antibody levels at lung and nasal mucosal sites, but also at distant mucosal sites such as the vaginal mucosal tissue. Mice immunized IN with BLP-adjuvanted vaccine and IM with non-adjuvanted vaccine were protected against weight loss upon homologous infection with H1N1 A/PR/8/34. Full protection against weight loss upon heterologous challenge with H1N1 A/PR/8/34 was seen in mice immunized IN with BLP-adjuvanted H1N1 A/New Caledonia-derived split virus vaccine, but not in those receiving the split virus vaccine IM. Mice immunized IN with BLP-adjuvanted vaccine had significantly lower lung viral titers upon homologous and heterologous challenge when compared to titers detected in mice immunized by IM injection of non-adjuvanted vaccine. Thus, adjuvantation of IN-administered influenza vaccines with BLPs effectively enhances systemic and local antibody responses leading to a superior protection against homologous and heterologous influenza infection compared to conventional IM immunization.     

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.     

Induction of opsonophagocytic killing activity with pneumococcal conjugate vaccine in human immunodeficiency virus-infected Ugandan adults

The levels of IgG determined by ELISA may have limited relevance in human immunodeficiency virus (HIV)-infected adults because of non-functional antibodies. 58 HIV-1-infected and 29 HIV-uninfected Ugandan adults were immunized with conjugate vaccine (CV) followed by polysaccharide vaccine (PV) after a 2-month interval, and the opsonophagocytic killing (OPK) titers against serotype 4 or 14 pneumococcal strains as well as the levels of serotype-specific IgG in sera were determined. Significant increases were found in the OPK titers and IgG levels for both serotypes after CV vaccination irrespective of HIV status. Increases in IgG levels and OPK titers were largely dependent on the CD4(+) cell counts, except for increases in the IgG levels for serotype 4. The proportions with serum OPK titer equal to or greater than 8 were 0-4.3% for serotype 4 and 26.7-42.9% for serotype 14 before vaccination, but the proportions increased up to 43.3-86.2% for serotype 4 and 63.3-96.6% for serotype 14 in all three groups 2 months after CV vaccination. The serum OPK titers remained at levels higher than the pre-vaccination level for at least 8 months after CV vaccination. A single dose of CV could afford some protective immunity in HIV-infected African adults before the introduction of antiretroviral therapy.     

Impact of prior flavivirus vaccination on immunogenicity and efficacy of an inactivated Zika vaccine in Indian rhesus macaques

Flaviviruses are antigenically related. We evaluated the immunogenicity and efficacy of Takeda’s purified inactivated Zika vaccine (PIZV) candidate in macaques previously vaccinated with several commercially available heterologous flavivirus vaccines. Heterologous flavivirus vaccination did not elicit Zika virus (ZIKV) neutralizing antibodies and did not impact neutralizing antibody titers after one dose of PIZV. After a second PIZV dose previous vaccination with flavivirus vaccines had variable impact on ZIKV neutralizing antibody titers. However, all macaques were protected against viremia after Zika virus challenge 8–12 months post-PIZV vaccination. Therefore, vaccine-induced immunity against heterologous flavivirus vaccines does not impact PIZV efficacy in macaques.     

LEE-encoded regulator (Ler) mutants elicit serotype-specific protection, but not cross protection, against attaching and effacing E. coli strains

We previously showed that single dose orogastric immunization with an attenuated regulatory Lee-encoded regulator (ler) mutant of the rabbit enteropathogenic Escherichia coli (REPEC) strain E22 (O103:H2) protected rabbits from fatal infection with the highly virulent parent strain. In the current study we assessed the degree of homologous (serotype-specific) and heterologous (cross-serotype) protection induced by immunization with REPEC ler mutant strains of differing serotypes, or with a prototype strain RDEC-1 (O15:H-) which expresses a full array of ler up-regulated proteins. We constructed an additional ler mutant using RDEC-1 thus, permitting immunization with a ler mutant of either serotype, O15 or O103, followed by challenge with a virulent REPEC strain of the same or different serotypes. Consistent with our previous data, the current study demonstrated that rabbits immunized with a RDEC-1 ler mutant were protected from challenge with virulent RDEC-H19A (RDEC-1 transduced with Shiga toxin-producing phage H19A) of the same serotype. Rabbits immunized with RDEC-1 or E22 derivative ler mutants demonstrated significant increase in serum antibody titers to the respective whole bacterial cells expressing O antigen but not to the LEE-encoded proteins. However, immunization with the ler mutants of either E22 or RDEC-1 failed to protect rabbits from infections with virulent organisms belonging to different serotypes. In contrast, rabbits immunized with the prototype RDEC-1 were cross protected against challenge with the heterologous E22 strain as shown by normal weight gain, and the absence of clinical signs of disease or characteristic attaching and effacing (A/E) lesions. Immunization with RDEC-1 induced significantly elevated serum IgG titers to LEE-encoded proteins. We thus, demonstrated homologous protection induced by the REPEC ler mutants and heterologous protection by RDEC-1. The observed correlation between elevated immune responses to the LEE-encoded proteins and the protection against challenge with heterologous virulent REPEC strain suggests that serotype-non-specific cross protection requires the expression of, and induction of antibody to, LEE-encoded virulence factors.     

Lactogenic antibody responses in cows vaccinated with recombinant bovine rotavirus-like particles (VLPs) of two serotypes or inactivated bovine rotavirus vaccines.

Triple-layered virus-like particles (VLPs) were produced in a baculovirus expression system from the two prevalent bovine rotavirus (BRV) serotypes, IND (P[5]G6) and 2292B (P[11]G10). Five groups of pregnant cows were inoculated intramuscularly and intramammarily with IND VLPs [BRV RF VP2, and IND VP4, 6, and 7, 250 microg per dose], 2292B VLPs [RF VP2, Cr VP4 (P[11]), and 2292B VP6 and 7, 250 microg per dose], combined IND/2292B VLPs (125 microg each VLP per dose), inactivated IND BRV (5x10(7)PFU per dose, pre-inactivation), or cell supernatant (mock-controls) in incomplete Freund's adjuvant. Serum, colostrum and milk were collected and tested for isotype-specific antibodies, and homologous and heterologous neutralizing antibodies (VN) to BRV by ELISA and VN tests, respectively. After vaccination, the IgG1 and homologous VN geometric mean antibody titers (GMTs) to BRV in serum of vaccinated groups were significantly (P<0.05) higher than in the mock-controls through postpartum day (PPD) 30. In colostrum, the IgG1 and IgA, and the homologous and heterologous VN GMTs of the IND VLP, 2292B VLP, combined IND/2292B VLP and the inactivated IND groups were significantly enhanced compared to the mock-controls, except for the heterologous VN GMTs in the inactivated IND group. However, the VLP vaccine groups had significantly higher homologous and heterologous VN GMTs than the inactivated IND group. The VN GMTs of the IND/2292B VLP group were statistically similar to the homologous VN GMTs of the IND or 2292B VLP groups, although the IgG1 GMT was lower. In milk, the IgG1 and homologous VN GMTs of the VLP groups were significantly higher than the inactivated IND or the mock-control groups through PPD30. However, the heterologous and homologous VN GMTs of inactivated IND group were statistically similar to the mock-control group at PPD0 and 30, respectively. These results demonstrate that the BRV antibody titers in serum, colostrum and milk are significantly enhanced by the use of triple-layered VLPs and inactivated IND vaccines, but significantly higher antibody responses were observed in the VLP vaccinated cows. The combined IND/2292B VLP vaccine induced comparable VN responses to BRV in serum, colostrum and milk compared to those induced by the individual IND or 2292B VLP vaccines, suggesting that at least two different serotypes can be mixed to confer maximum antibody responses to the incorporated serotypes.     

Strategies for inducing protection against avian influenza A virus subtypes with DNA vaccines

The cross-species transfer of a H5N1 influenza virus from birds to humans, and the systemic spread of this virus in mice, has accelerated the efforts to devise protective strategies against lethal influenza viruses. DNA vaccination with the highly conserved nucleoprotein gene appears to provide cross protection against influenza A viruses in murine models. Whether such vaccines would protect human hosts against different influenza A viruses, including strains with pandemic potential, is unclear. Our aim in this study is to evaluate the ability of a combination DNA vaccine consisting of two plasmids encoding the HA genes from two different subtypes and a DNA vaccine encoding the viral nucleoprotein gene from a H5 virus to induce protection against highly lethal infection caused by H5 and H7 influenza viruses in chickens. Chickens given a single dose of plasmids expressing H5 and H7 hemagglutinins protected the birds from infection by either subtype. However, birds immunized with nucleoprotein DNA and challenged with either A/Ck/Vic/1/85(H7N7) or A/Ty/Ir/1/83 (H5N8) showed definite signs of infection, suggesting inadequate immunity against viral infection. Fifty percent of the nucleoprotein DNA immunized birds survived infection by influenza A/Ty/Ir/1/83 (H5N8) virus (virus of same subtype) while 42% survived infection by influenza A/Ck/Vic/1/85/(H7N7) virus (virus of a different subtype). These studies demonstrate that immunization with DNA encoding a type-specific gene may not be effective against either homologous or heterologous strains of virus, particularly if the challenge virus causes a highly lethal infection. However, the combination of HA subtype vaccines are effective against lethal infection caused by viruses expressing any of the HA subtypes used in the combination preparation.     

Protection with recombinant Clostridium botulinum C1 and D binding domain subunit (Hc) vaccines against C and D neurotoxins

Recombinant botulinum Hc (rBoNT Hc) vaccines for serotypes C1 and D were produced in the yeast Pichia pastoris and used to determine protection against four distinct BoNT C and D toxin subtypes. Mice were vaccinated with rBoNT/C1 Hc, rBoNT/D Hc, or with a combination of both vaccines and challenged with BoNT C1, D, C/D, or D/C toxin. Mice receiving monovalent vaccinations were partially or completely protected against homologous toxin and not protected against heterologous toxin. Bivalent vaccine candidates completely survived challenges from all toxins except D/C toxin. These results indicate the recombinant C1 and D Hc vaccines are not only effective in a monovalent formula but offer complete protection against both parental and C/D mosaic toxin and partial protection against D/C mosaic toxin when delivered as a bivalent vaccine.     

Sustained functional serotype-specific antibody after primary and secondary vaccinations with a pneumococcal polysaccharide vaccine in elderly patients with chronic lung disease

An observational study was conducted to determine immunogenicity before and after primary and secondary vaccinations with 23-valent pneumococcal polysaccharide vaccine in a cohort of 40 elderly patients with chronic lung diseases. Safety of this vaccine was also compared between primary and secondary vaccination. We analyzed serotype-specific immunoglobulin G (IgG) and the opsonization index (OI) for serotypes 6B, 14, 19F, and 23F and compared adverse local and systemic reactions. The levels of serotype-specific IgG and the OIs significantly increased 1 month after primary and secondary vaccinations. Peak levels of IgG after secondary vaccination were 5–20% lower than those after primary vaccination, while serotype-specific OIs after secondary vaccination were comparable with those after primary vaccination. The levels of serotype-specific IgG required for 50% killing significantly decreased 1 month after vaccination. These values for serotypes 14, 19F, and 23F were slightly elevated immediately before secondary vaccination, but those for serotype 6B did not change. After secondary vaccination, these values declined slightly for serotypes 14, 19F, and 23F and remained low for serotype 6B. Although self-limited local and systemic reactions were more frequent after secondary vaccination compared with primary vaccination, no serious systemic reaction was found after either vaccination. Our data suggest a sustained functional serotype-specific IgG after primary and secondary vaccination and confirmed the safety of secondary vaccination among elderly individuals with chronic lung disease.     

Memory B cell response to a PCV-13 booster in 3.5 year old children primed with either PCV-7 or PCV-13

Pneumococcal protein-polysaccharide conjugate vaccines provide direct protection against Streptococcus pneumoniae through the induction of persistent anti-polysaccharide antibodies, and by priming for a rapid secondary antibody response. Memory B cells (B_MEM) generated during an initial immune response are responsible for both the more rapid and quantitatively greater secondary antibody response and are also thought to contribute to the ongoing production of plasma cells providing long-term antibody persistence. We recruited 3.5-year-old children who had participated in a previous clinical trial comparing infant immunization with either a 7-valent (PCV-7) or a 13-valent pneumococcal conjugate vaccine (PCV-13) to investigate whether prior priming with pneumococcal antigens influences B_MEM responses. Blood was taken before and 1 month after a PCV-13 booster. B_MEM were quantified using a cultured ELISpot assay for pneumococcal serotypes 1, 3, 4, 14, 19A, 23F, and with diphtheria and tetanus toxoid as controls, and then correlated with serotype-specific IgG concentrations and opsonophagocytic activity (OPA) titers. In total, blood samples from 62 participants were available for analysis. Serotype-specific B_MEM frequencies were generally low at baseline (before boost) although for serotypes 14 and 3, they were significantly higher in children primed with PCV-13 than PCV-7 primed children. Following the PCV-13 booster, B_MEM frequencies increased and were not different between the groups for all serotypes. A strong inverse correlation was found between antibody concentrations and OPA titers at baseline and B_MEM following booster vaccination for serotype 3 but not for other serotypes suggesting that, for this serotype, pre-existing serotype-specific antibodies may inhibit B_MEM formation in response to vaccination.Clinicaltrials.gov registration number: NCT01095471.