Vicki Barniak

Vaccine potential of the Neisseria meningitidis 2086 lipoprotein.

ABSTRACT A novel antigen that induces cross-reactive bactericidal antibodies against a number of Neisseria meningitidis strains is described. This antigen, a ∼28-kDa lipoprotein called LP2086, was first observed within a complex mixture of soluble outer membrane proteins (sOMPs) following a series of fractionation, protein purification, and proteomics steps. Approximately 95 different neisserial isolates tested positive by Western blotting and PCR screening methods for the presence of the protein and the gene encoding LP2086. The strains tested included isolates of N. meningitidis serogroups A, B, C, W135, and Y, Neisseria gonorrhoeae, and Neisseria lactamica. To better understand the microheterogeneity of this protein, the 2086 genes from 63 neisserial isolates were sequenced. Two different subfamilies of LP2086 were identified based on deduced amino acid sequence homology. A high degree of amino acid sequence similarity exists within each 2086 subfamily. The highest degree of genetic diversity was seen between the two subfamilies which share approximately 60 to 75% homology at the nucleic acid level. Flow cytometry (fluorescence-activated cell sorting) analyses and electron microscopy indicated that the LP2086 is localized on the outer surface of N. meningitidis. Antiserum produced against a single protein variant was capable of eliciting bactericidal activity against strains expressing different serosubtype antigens. Combining one recombinant lipidated 2086 (rLP2086) variant from each subfamily with two rPorA variants elicited bactericidal activity against all strains tested. The rLP2086 family of antigens are candidates worthy of further vaccine development.

Recombinant PhpA Protein, a Unique Histidine Motif-Containing Protein from Streptococcus pneumoniae, Protects Mice against Intranasal Pneumococcal Challenge

ABSTRACT The multivalent pneumococcal conjugate vaccine is effective against both systemic disease and otitis media caused by serotypes contained in the vaccine. However, serotypes not covered by the current conjugate vaccine may still cause pneumococcal disease. To address these serotypes and the remaining otitis media due to Streptococcus pneumoniae, we have been evaluating antigenically conserved proteins from S. pneumoniae as vaccine candidates. A previous report identified a 20-kDa protein with putative human complement C3-proteolytic activity. By utilizing the publicly released pneumococcal genomic sequences, we found the gene encoding the 20-kDa protein to be part of a putative open reading frame of approximately 2,400 bp. We recombinantly expressed a 79-kDa fragment (rPhpA-79) that contains a repeated HxxHxH motif and evaluated it for vaccine potential. The antibodies elicited by the purified rPhpA-79 protein were cross-reactive to proteins from multiple strains of S. pneumoniae and were against surface-exposed epitopes. Immunization with rPhpA-79 protein adjuvanted with monophosphoryl lipid A (for subcutaneous immunization) or a mutant cholera toxin, CT-E29H (for intranasal immunization), protected CBA/N mice against death and bacteremia, as well as reduced nasopharyngeal colonization, following intranasal challenge with a heterologous pneumococcal strain. In contrast, immunization with the 20-kDa portion of the PhpA protein did not protect mice. These results suggest that rPhpA-79 is a potential candidate for use as a vaccine against pneumococcal systemic disease and otitis media.

Heterotypic protection from rotavirus infection in mice vaccinated with virus-like particles.

Virus-like particles (VLPs) composed of rotavirus VP2, VP6, and VP7 of G1 or G3 serotype specificity were produced in insect cells coinfected with recombinant baculoviruses expressing single rotavirus genes. The VLPs were purified and subsequently evaluated for immunogenicity and protection in the adult mouse model of rotavirus infection. Mice were vaccinated twice intramuscularly with G1 VLPs formulated with Quillaja saponaria (QS-21) or adsorbed to aluminium hydroxide (AlOH), or with G1 VLPs alone. G3 VLPs, G1 plus G3 VLPs, inactivated SA11 virions formulated with QS-21, or adjuvants were similarly inoculated as controls. Mice were examined for serum and fecal antibody responses by ELISA or microneutralization assays. Protective efficacy of the VLP vaccine formulations against oral challenge with the G3 murine ECwt rotavirus was assessed by comparing the antigen shed in stool of the VLP-vaccinated mice to that of the adjuvant-immunized mice. G1 VLPs in QS-21 induced significantly higher serum and intestinal antibody titers than G1 VLPs in AlOH or G1 VLPs alone. QS-21 also heightened serum and fecal antibody responses to G3 VLPs. These QS-21-augmented antibody responses were further characterized by equivalent IgG1 and IgG2a titers in sera, suggesting that G1 or G3 VLPs in QS-21 induced a balanced Th1/Th2 response. G1 VLPs in QS-21 induced partial protection (88%) against oral challenge with the heterotypic ECwt virus, whereas G3 VLPs in QS-21 induced complete protection (100%). In contrast, G1 VLPs when formulated with AlOH induced a predominant Th2 response and did not protect (1%) mice from virus challenge. Our results indicate that the type of adjuvant used clearly influences both antibody responses to rotavirus VLPs and the protective efficacy against rotavirus infections. These data have important implications for the development of parenteral vaccines to ameliorate rotavirus disease.

Biochemical and immunologic comparison of virus-like particles for a rotavirus subunit vaccine.

A parenterally administered rotavirus vaccine composed of virus-like particles (VLPs) is being evaluated for human use. VLPs composed of bovine VP6 and simian VP7 (SA11, G3) proteins (6/7-VLPs) or of bovine VP2, bovine VP6, and simian VP7 (SA11, G3) proteins (2/6/7-VLPs) were synthesized and purified from Sf9 insect cells co-infected with recombinant baculoviruses. 6/7- and 2/6/7-VLP administered parenterally (i.m.) in mice had comparable immunogenicity, but the 2/6/7-VLPs were more homogeneous and stable. The inclusion of the VP2 capsid contributed to particle formation and stability. The adjuvant QS-21 significantly enhanced the immunogenicity of 2/6/7-VLPs over A10H or saline alone. Equivalent serum neutralizing antibody responses were induced over the range of 1-15 microg/dose of 2/6/7-VLPs administered with the range of 5-20 microg/dose of QS-21. The immunogenicity of 2/6/7-VLPs and inactivated SA11 virus were comparable. 2/6/7-VLPs are a promising candidate for a parenterally delivered rotavirus subunit vaccine.

PppA, a Surface-Exposed Protein of Streptococcus pneumoniae, Elicits Cross-Reactive Antibodies That Reduce Colonization in a Murine Intranasal Immunization and Challenge Model

ABSTRACT The multivalent pneumococcal conjugate vaccine is effective against both systemic disease and otitis media caused by serotypes contained in the vaccine. However, serotypes not covered by the present conjugate vaccine may still cause pneumococcal disease. To address these serotypes, and the remaining otitis media due to Streptococcus pneumoniae, efforts have been devoted to identifying protective protein antigens. Immunity to conserved surface proteins important for adhesion, nutrient acquisition, or other functions could result in a reduction of colonization and a lower disease potential. We have been searching for conserved surface-exposed proteins from S. pneumoniae that may be involved in pathogenesis to test as vaccine candidates. Here, an ∼20-kDa protein that has significant homology to a nonheme iron-containing ferritin protein from Listeria innocua and other bactoferritins was identified as pneumococcal protective protein A (PppA). We expressed and purified recombinant PppA (rPppA) and evaluated its potential as a vaccine candidate. The antibodies elicited by purified rPppA were cross-reactive with PppA from multiple strains of S. pneumoniae and were directed against surface-exposed epitopes. Intranasal immunization of BALB/c mice with PppA protein and either a synthetic monophosphoryl lipid A analog, RC529AF, or a cholera toxin mutant, CT-E29H, used as an adjuvant reduced nasopharyngeal colonization in mice following intranasal challenge with a heterologous pneumococcal strain. PppA-specific systemic and local immunoglobulin G (IgG) and IgA antibody responses were induced. The antisera reacted with whole cells of a heterologous S. pneumoniae type 3 strain. These observations indicate that PppA may be a promising candidate for inclusion in a vaccine against pneumococcal otitis media.

Evaluation of Recombinant Lipidated P2086 Protein as a Vaccine Candidate for Group B Neisseria meningitidis in a Murine Nasal Challenge Model

ABSTRACT Neisseria meningitidis is a major causative agent of bacterial meningitis in human beings, especially among young children (≤2 years of age). Prevention of group B meningococcal disease represents a particularly difficult challenge in vaccine development, due to the inadequate immune response elicited against type B capsular polysaccharide. We have established an adult mouse intranasal challenge model for group B N. meningitidis to evaluate potential vaccine candidates through active immunization. Swiss Webster mice were inoculated intranasally with meningococci, and bacteria were recovered from the noses for at least 3 days postchallenge. Iron dextran was required in the bacterial inoculum to ensure sufficient meningococcal recovery from nasal tissue postchallenge. This model has been utilized to evaluate the potential of a recombinant lipidated group B meningococcal outer membrane protein P2086 (rLP2086) as a vaccine candidate. In this study, mice were immunized subcutaneously with purified rLP2086 formulated with or without an attenuated cholera toxin as an adjuvant. The mice were then challenged intranasally with N. meningitidis strain H355 or M982, and the colonization of nasal tissue was determined by quantitative culture 24 h postchallenge. We demonstrated that immunization with rLP2086 significantly reduced nasal colonization of mice challenged with the two different strains of group B N. meningitidis. Mice immunized with rLP2086 produced a strong systemic immunoglobulin G response, and the serum antibodies were cross-reactive with heterologous strains of group B N. meningitidis. The antibodies have functional activity against heterologous N. meningitidis strain, as demonstrated via bactericidal and infant rat protection assays. These results suggest that rLP2086 is a potential vaccine candidate for group B N. meningitidis.

Evaluation of a 74-kDa transferrin-binding protein from Moraxella (Branhamella) catarrhalis as a vaccine candidate.

An outer membrane protein from Moraxella catarrhalis with a mass of 74-kDa was isolated and evaluated as a vaccine candidate. The 74-kDa protein binds transferrin, and appears to be related to the other proteins from the organism that are reported to bind transferrin. The 74-kDa protein possessed conserved epitopes exposed on the bacterial surface. This is based on the reactivity with whole bacterial cells as well as complement dependent bactericidal activity of sera from mice immunized with the isolated proteins from the O35E and TTA24 isolates. However, there was divergence in the degree of antibody cross-reactivity with the protein from one strain to another. This serotypic divergence was reflected in both the complement-dependent bactericidal activities of the antibodies elicited in mice and the capacity of immune mice to clear the bacteria in a murine pulmonary model. Antibodies affinity purified from human plasma lacked bactericidal activity even though they were reactive with all the tested isolates. The 74-kDa protein appears to be a good vaccine candidate, but more studies are needed to understand its antigenic variability and whether antibodies toward it are protective.

Passive immunization with Neisseria meningitidis PorA specific immune sera reduces nasopharyngeal colonization of group B meningococcus in an infant rat nasal challenge model.

To examine the protective efficacy of specific immune sera generated by meningococcal vaccine candidates against nasopharyngeal colonization, we developed an infant rat nasal colonization model for group B meningococcus. In this model, Sprague-Dawley infant rats were challenged intranasally in with host adapted, piliated Neisseria meningitidis group B strains H355 or H44/76 administered concurrently with iron dextran. Colonization was assessed by quantitative culture of nasal homogenates and expressed as log(10) colony forming units (c.f.u.) per nose. Three to five log(10) c.f.u. of N. meningitidis were routinely recovered from the nasal tissue up to 4 days post-challenge. Passive immunization (i.p.) of the infant rats with either PorA or whole cell antisera 24 h prior to homologous challenge resulted in a significant reduction of N. meningitidis colonization in the nasal tissues of these animals. These results demonstrate that this model can be utilized to evaluate the role of antibody to prevent the initial nasopharyngeal colonization by group B meningococcus.

Correction for Fletcher et al., “Vaccine Potential of the Neisseria meningitidis 2086 Lipoprotein”

[This corrects the article DOI: 10.1128/IAI.72.4.2088-2100.2004.].