Strains SPEC 6B (6B OPKAserotype 6B, PspA3, resistant to spectinomycin) and EMC 23F (23F OPKACserotype 23F, PspA 2) were kindly provided by Dr Moon Nahm (University of Alabama at Birmingham, USA)
August 1, 2022
Strains SPEC 6B (6B OPKAserotype 6B, PspA3, resistant to spectinomycin) and EMC 23F (23F OPKACserotype 23F, PspA 2) were kindly provided by Dr Moon Nahm (University of Alabama at Birmingham, USA). Recombinant Proteins and Vaccine Formulations The N-terminal fragments of PspA from clades 1, 2, 3 and 4 (from strains St 435/96, St371/00, St259/98 and St255/00, respectively) were expressed in BL21 SI (Invitrogen) and purified by metal affinity chromatography as previously described . if immunization with PspA would be able to control co-colonization. We evaluated nasal immunization with recombinant PspA (rPspA) in a model of co-colonization with two Rabbit polyclonal to AGO2 strains expressing different PspAs. Mice were immunized intranasally with rPspAs from clades 1 to 4 (rPspA1, rPspA2, rPspA3 or rPspA4) using whole-cell pertussis vaccine (wP) as adjuvant. Mice were then challenged with a mixture of two serotype 6B isolates St491/00 (PspA1) and St472/96 (PspA4). Immunization with rPspA1+wP and rPspA4+wP reduced colonization with both strains and the mixture of rPspA1+rPspA4+wP induced greater reduction than a single antigen. Immunization rPspA1+rPspA4+wP also reduced colonization when challenge experiments were performed with a mixture of isolates of serotypes 6B (PspA3) and 23F (PspA2). Furthermore, none of the tested formulations led to a pronounced increase in colonization of one isolate over the other, showing that this vaccine strategy would not favor replacement. Interestingly, the adjuvant wP by itself already led to some reduction in pneumococcal colonization, indicating the induction of non-specific immune responses. Anti-rPspA IgG was observed in serum, nasal wash (NW) and bronchoalveolar lavage fluid (BALF) samples, whereas animals inoculated with AN7973 formulations made up of the adjuvant wP (with or without rPspA) showed higher levels of IL-6 and KC in NW and increase in tissue macrophages, B cells and CD4+T cells in BALF. Introduction is usually part of the nasopharyngeal microbiota of healthy humans, maintaining a commensal relationship with the host. However, it can cause several diseases with high mortality and morbidity, such as meningitis, bacteremia and pneumonia, and other common respiratory tract infections such as otitis media and sinusitis. Colonization of the nasopharynx is usually a prerequisite for pneumococcal disease development and transmission of bacteria. Colonization rates vary according to geographical location and AN7973 socioeconomic conditions, with prevailing rates of 20C90% in children less than five years of age, and 1C10% in adults [1C4]. Simultaneous colonization by multiple pneumococcal strains is also common and up to 50% of colonized children carry simultaneously two or more strains of [5C8]. The currently available vaccines are based on the response against the capsular polysaccharide (PS) and PS-conjugate vaccines (PCVs) show high efficacy against invasive pneumococcal disease (IPD) caused by vaccine serotypes. Though a net reduction in pneumococcal disease was observed in most countries, the widespread use of PCVs led to the substitution of prevalent serotypes, with an increase in incidence of non-vaccine serotype strains both in colonization [2,9] and IPD [10C13]. This phenomenon of serotype replacement is an example of the effect of the selective pressure promoted by vaccines that do not have complete coverage against all variants of the pathogen. Protein antigens are being studied as alternative vaccines against pneumococcal disease and a promising candidate is usually Pneumococcal surface protein A (PspA). Mature PspA is composed of a domain name at the C-terminal region that anchors the protein to the cell surface through conversation with choline residues of teichoic and lipoteichoic acids. This domain name is usually followed by AN7973 a central proline-rich domain name and an N-terminal -helical component exposed around the bacterial surface . PspA shows variability in different isolates and sequence-based classification divide PspA variants AN7973 into three families, that are further subdivided into six clades: family 1 (clades 1 and 2), family 2 (clades 3, 4 and 5) and family 3 (clade 6) . To achieve complete coverage, it was suggested that a PspA-based vaccine should contain at least one PspA from each of the two major families (1 and 2) . Our group has previously shown that parenteral immunization of mice with a recombinant PspA from clade 4 (rPspA4, family 2) or from clade 5 (rPspA5, family 2) induces protection against lethal pneumococcal challenge with strains expressing PspA from families 1 and 2 . Furthermore, the use of the whole-cell pertussis vaccine (wP) as adjuvant for nasal immunization of mice with rPspA5 induced protection against a lethal intranasal challenge model and also against nasal colonization with a pneumococcal strain expressing PspA from family 1. Studies that analyzed the genomes of 240 pneumococcal strains from a multidrug resistant lineage  and the genomes of 616 strains isolated from the nasopharynx from healthy individuals after the introduction of the heptavalent conjugate pneumococcal vaccine (PCV7)  showed that besides loci encoding a putative genetic element and the loci involved in capsule synthesis, the genes under selective pressure with the highest recombination rates were and strains were produced in Todd-Hewitt broth (Difco) supplemented with 0.5% yeast extract (THY) at 37C without shaking. Bacteria were usually plated in blood agar and produced overnight at 37C before inoculation in THY. Stocks were maintained at ?80C in THY containing 20% glycerol. Strains St491/00 (serotype 6B, PspA1) and St472/96 (serotype 6B, PspA4, resistant to trimethoprim) were kindly provided by Dr Maria Cristina Brandileone (Instituto Adolfo Lutz, S?o Paulo, Brazil). Strains SPEC 6B (6B OPKAserotype 6B, PspA3, resistant to.