Protection against experimental infection with group B streptococcus by immunization with a bivalent protein vaccine

doi:10.1016/S0264-410X(98)00218-7

Vaccine

Volume 17, Issue 5, February 1999, Pages 454-458

https://doi.org/10.1016/S0264-410X(98)00218-7 Get rights and content

Abstract

Group B streptococcus (GBS), a bacterium with polysaccharide capsule, is the major cause of sepsis and meningitis in early infancy. Recent work has indicated that immunity to GBS infection can be elicited by the surface proteins Rib and α, either of which is expressed by most GBS strains causing invasive infections. Here we show that a bivalent vaccine, composed of purified Rib and α mixed with aluminum hydroxide (alum), an adjuvant accepted for human use, elicits an antibody response to each of the two antigens. Moreover, the bivalent vaccine was found to protect against experimental infection with GBS strains representing the four classical serotypes. Our results represent an encouraging step towards the development of a human GBS vaccine based on pure protein antigens.

Introduction

Group B streptococci (GBS) are the leading cause of life threatening bacterial infections, e.g. pneumonia, sepsis and meningitis, during the neonatal period. In the United States there are about 10 000 cases of invasive GBS infections per year with a mortality of ∼10% and many of the infants that survive meningitis may suffer from neurologic sequelae[1]. In addition, GBS has emerged as an important pathogen among nonpregnant adults, especially the elderly or patients with chronic underlying diseases[2]. This situation makes it of interest to develop a GBS vaccine suitable for human use3, 4.

All strains of GBS express a polysaccharide capsule, with nine capsular serotypes identified so far[5]. Strains of the four classical serotypes, Ia, Ib, II and III, are responsible for the large majority of invasive neonatal infections[1]. Strains of type III are of particular interest since they have been reported to account for two thirds of GBS infections in neonates and approximately 90% of cases of neonatal meningitis[1]. Since the capsule is a virulence factor that confers protective immunity, efforts are under way to develop a multivalent conjugate vaccine based on polysaccharides6, 7.

Cell surface proteins of GBS have also attracted attention as possible vaccine candidates since they confer protective immunity in animal models8, 9, 10, 11. Among the four classical serotypes of GBS, approximately 90% of all strains express either Rib and α, two members of a novel family of streptococcal cell surface proteins. These extremely repetitive proteins show extensive amino acid residue identity12, 13, but exhibit no or little immunological cross-reactivity9, 11. Since proteins have several advantages as vaccine components, it is of interest to evaluate Rib and α as components in a GBS vaccine suitable for human use.

We have previously demonstrated that active immunization of mice with highly purified preparations of Rib or α, mixed with Freund's adjuvant, conferred protection against lethal infection with strains expressing the corresponding antigen[11]. This result encouraged us to study the properties of a bivalent protein vaccine based on purified Rib and α mixed with alum, an adjuvant commonly used in human vaccines[14]. The results reported here demonstrate that this bivalent vaccine elicited an antibody response to both protein antigens, Rib and α, and conferred protection against experimental infection with GBS strains representing each of the four classical serotypes.

Section snippets

Bacterial strains

Four GBS strains representing the four classical capsular serotypes were used for purification of proteins and/or protection studies: A909, type Ia[12]; SB35sed1, type Ib[9]; 118/158, type II[11]; and BM110, type III9, 11. Bacterial strains were grown in Todd–Hewitt broth.

Preparation of protein vaccines

The Rib and α proteins were isolated from streptococci by several purification steps, to ensure that they were free of contaminating polysaccharides, as described previously9, 11. The Rib protein was purified from the type III

Antibody response in mice immunized with monovalent or bivalent vaccines

The studies described here were performed with highly purified preparations of the Rib and α proteins, and a combination of these two proteins (Fig. 1A). The Rib and α proteins did not show any detectable immunological cross-reactivity when analyzed in Western blots with mouse antisera, in agreement with previously reported experiments employing rabbit antisera9, 11.

Immunization of mice with one of the Rib or α proteins, mixed with Freund's adjuvant, was previously shown to induce an IgG

Discussion

Many life-threatening infections in humans are caused by bacteria with polysaccharide capsule. Efforts to develop polysaccharide vaccines against such infections have, in several cases, met with difficulties, e.g. poor immunogenicity of the antigens and immunological cross-reactivity with human tissue. Therefore, it is of interest to analyze the possibility of using bacterial surface proteins as vaccines against these infections. Indeed, protein antigens may have several advantages: most

Acknowledgements

We are grateful to Dr. I. Krantz for valuable advice on statistics and to Mrs. U. Regnér for technical assistance. This work was supported by The Swedish Medical Research Council (grant 9490), the Medical Faculty of Lund University, SmithKline Beecham Biologicals s.a., the Alfred Österlund Trust, the Crafoord Trust, the Johan and Greta Kock Trust, King Gustaf V's 80th Birthday Trust, the Magnus Bergvall Trust and the `Förenade liv' Mutual Group Life Insurance Company, Stockholm.

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Group B streptococcus and respiratory syncytial virus are leading causes of infant morbidity and mortality worldwide. No licensed vaccines are available for either disease, but vaccines for both are under development. Severe respiratory syncytial virus disease can be prevented by passively administered antibody. The presence of maternal IgG antibody specific to respiratory syncytial virus is associated with reduced prevalence and severity of respiratory syncytial virus disease in the first few weeks of life, whereas maternal serotype-specific anticapsular antibody is associated with protection against both early-onset and late-onset group B streptococcus disease. Therefore, vaccination in pregnancy might protect infants against both diseases. This report describes what is known about immune protection against group B streptococcus and respiratory syncytial virus, identifies knowledge gaps regarding the immunobiology of both diseases, and aims to prioritise research directions in maternal immunisation.
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There are a number of obvious advantages with such an approach but until very recently none had entered clinical trials in humans. MinervaX, a privately held Danish biotech company, has recently initiated a Phase I clinical trial with a protein-only vaccine based on a fusion of two immunogenic and protective protein domains from selected surface proteins of GBS (N-terminals of AlphaC and Rib) [35,36]. These proteins appear to have a broad distribution among GBS isolates.
Streptococcus agalactiae (group B streptococcus (GBS)) is the leading cause of neonatal sepsis and meningitis in many countries. Intrapartum antibiotic strategies have reduced the incidence of early-onset neonatal GBS in a number of countries but have had no impact on late onset GBS infection (LOD). In low/middle income settings, the disease burden remains uncertain although in several countries of Southern Africa appears comparable to or higher than that of high-income countries. As disease may be rapidly fulminating cases can be missed before appropriate samples are obtained and this may lead to underestimation of the true burden. Given the rapid onset and progression within hours of birth as well as the deficiencies in IAP strategies and absence of a solution for preventing LOD, it is clear that administration of a suitable vaccine in pregnancy could provide a better solution in all settings; it should also be cost effective. The current leading vaccine candidates are CPS-protein conjugate vaccines but protein-based vaccines are also in development and one has recently commenced clinical trials.
Immunological fingerprinting of group B streptococci: From circulating human antibodies to protective antigens
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The best characterized are the host extracellular matrix binding proteins, such as the epithelial cell adhesin Rib [9], the Fibrinogen-binding protein A (FbsA) [10,11], the fibronectin-binding, cellular invasion and complement inactivating protein C5a peptidase (ScpB) (reviewed in [12]), the leucine-rich repeat containing adhesin/invasin LrrG [13] and the adhesin and complement inhibiting BibA [14]. Several of the surface proteins with defined roles in GBS pathogenesis have been identified as protective antigens in mice: the Sip protein [15,16], C5a peptidase [17], LrrG [13], Rib, and Cα and β proteins [9,18,19]. Stimulated by the availability of complete genome sequences, novel and comprehensive approaches were recently applied to discover conserved GBS surface proteins.
Group B streptococcus is one of the most important pathogens in neonates, and causes invasive infections in non-pregnant adults with underlying diseases. Applying a genomic approach that relies on human antibodies we identified antigenic GBS proteins, among them most of the previously published protective antigens. In vitro analyses allowed the selection of conserved candidate antigens that were further evaluated in murine lethal sepsis models using several GBS strains. In active and passive immunization models, we identified four protective GBS antigens, FbsA and BibA, as well as two hypothetical proteins, all shown to contribute to virulence based on gene deletion mutants. These protective antigens have the potential to be components of novel vaccines or targets for passive immune prophylaxis against GBS disease.
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Streptococcus suis is an important swine and human pathogen, and also an emerging zoonotic agent. A surface-associated subtilisin-like serine protease (SspA) of S. suis was identified by screening a genomic expression library as fragments of this protein reacted most strongly with convalescent-phase pig sera. The sspA gene is present in 29 of 33 S. suis serotypes reference strains and is expressed on the surface of S. suis. Relative real-time quantitative PCR assay demonstrated that sspA mRNA expression in vivo was several thousand fold of that in vitro. A sspA⁻ mutant was generated from a S. suis serotype 2 strain SC19 by allelic exchange. The mutant was not different from the wild type strain in subcellular structures and in hemolytic phenotype. However, the virulence of the sspA⁻ mutant was markedly lower than the wild type in pigs as demonstrated in experimental infections. These data indicated that the surface-associated protein SspA is a conserved virulence factor of S. suis and is involved in the pathogenesis of S. suis.
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Therefore, we first asked whether the list of 43 proteins found on the surface of COH1 included the protective antigens so far described for GBS. Seven protective antigens have been reported so far (Table II): the C5a peptidase (SAN_1369) (17, 18), the Gro/surface immunogenic protein SIP (SAN_0040) (19, 20), the reticulocyte-binding protein/leucine-rich repeat protein (LrrG) (SAN_0509) (21), the protein Rib (22–24), and the three pilus variants PI-1, PI-2a, and PI-2b (6, 25, 26). The genes encoding these protective antigens are all present in the COH1 genome with the exception of the PI-2a pilus island; therefore six of the seven protective antigens were expected to be found on the surface of COH1.
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Protection against experimental infection with group B streptococcus by immunization with a bivalent protein vaccine

Abstract

Introduction

Section snippets

Bacterial strains

Preparation of protein vaccines

Antibody response in mice immunized with monovalent or bivalent vaccines

Discussion

Acknowledgements

J Biol Chem

J Biol Chem

Vaccine

Vaccine

A population-based assessment of invasive disease due to group B Streptococcus in nonpregnant adults

N Engl J Med

Immunization to prevent group B streptococcal disease: victories and vexations

J Infect Dis

Comparison of prevention strategies for neonatal group B streptococcal infection. A population-based economic analysis

JAMA

Synthesis and immunological properties of conjugates composed of group B streptococcus type III capsular polysaccharide covalently bound to tetanus toxoid

Infect Immun