Med Microbiol Immunol (2015) 204:185–191 DOI 10.1007/s00430-014-0353-2
ORIGINAL INVESTIGATION
Vaccination with Streptococcus pyogenes nuclease A stimulates a high antibody response but no protective immunity in a mouse model of infection Fiona J. Radcliff · John D. Fraser · Thomas Proft
Received: 23 July 2014 / Accepted: 4 August 2014 / Published online: 15 August 2014 © Springer-Verlag Berlin Heidelberg 2014
Abstract Streptococcus pyogenes is a human pathogen which causes a spectrum of diseases ranging from pharyngitis to rheumatic fever, necrotising fasciitis and toxic shock syndrome. Development of a vaccine for S. pyogenes has been confounded both by the diversity of the diseasecausing serotypes and the spectre of inadvertently stimulating autoimmunity. The S. pyogenes nuclease A (SpnA) is a recently characterised virulence factor that is highly conserved across strains and expressed during human disease. Deletion of spnA from S. pyogenes results in reduced survival of bacteria in whole human blood and attenuated virulence in a mouse model of infection. Collectively these features suggest that SpnA has potential as a vaccine candidate for S. pyogenes. Mice vaccinated subcutaneously with single or multiple doses of recombinant SpnA emulsified in Incomplete Freund’s Adjuvant developed a robust and durable IgG response, including neutralising activity, to this protein. However, vaccination with rSpnA conferred no advantage in terms of lesion development, disease symptoms or colonisation levels after a sub-lethal subcutaneous challenge with S. pyogenes. Anti-SpnA serum IgG responses and neutralising activity were increased in response to challenge, indicating that SpnA is expressed in vivo. SpnA is unlikely to be a suitable antigen for a vaccine against S. pyogenes. Keywords Group A Streptococcus · Streptococcus pyogenes · Vaccination · Nuclease · Spy0747 · SpnA
F. J. Radcliff (*) · J. D. Fraser · T. Proft Department of Molecular Medicine and Pathology, School of Medical Sciences and Maurice Wilkins Centre for Biodiscovery, University of Auckland, Auckland 1142, New Zealand e-mail:
[email protected]
Introduction Streptococcus pyogenes (Group A Streptococcus; GAS) causes a broad spectrum of diseases ranging from pharyngitis or impetigo to scarlet fever and streptococcal toxic shock syndrome. Infection with GAS can lead to the development of autoimmune conditions such as rheumatic fever, acute glomerulonephritis and rheumatic heart disease. GAS remains a significant contributor to global morbidity and mortality that disproportionately affects individuals from developing nations as well as disadvantaged populations within developed nations [1, 2]. Penicillin is an effective treatment option for GAS, but does not prevent recurrence of infection or reduce the economic costs associated with symptomatic disease. Screening and prophylactic administration of penicillin are effective approaches for reducing cases of acute rheumatic fever in high risk communities [3], but this strategy is only feasible in regions where there is access to high standard healthcare. Development of a protective vaccine would be an effective way to control or eliminate GAS disease worldwide. However, there are a number of challenges associated with developing an effective vaccine for GAS including the diversity of unique serotypes, geographical variability in distribution of serotypes and the risk of stimulating hostreactive antibodies [4]. A number of vaccine candidate antigens have shown promise in pre-clinical models [4], but the only vaccine to enter a clinical trial in the last 30 years has been StreptAvax, a 26-valent vaccine based on the N-terminal of M protein [5]. This vaccine stimulated increased antibody responses to the vaccine antigens and patient serum showed enhanced opsonising activity in in vitro assays in human clinical trials, with no evidence of host tissue-reactive antibodies [6]. An improved 30-valent vaccine with broader
13
186
coverage of dominant M types in Europe and the United States of America is currently undergoing clinical trials [7, 8]. The success of these trials, particularly the safety profile, has re-invigorated the search for novel GAS vaccine candidate antigens. Advances in genomic and proteomic technologies have resulted in effective high-throughput screens to identify potential antigens primarily based on their immunogenicity and accessibility to the host immune response [9–12]. A vaccine against multiple conserved expressed epitopes would be highly desirable. The importance of extracellular nucleases in bacterial virulence was revealed after the discovery of neutrophil extracellular traps (NETs), structures containing granule and nuclear components that can disarm and kill bacteria [13]. Removal of the ability to produce major endonucleases has been demonstrated to reduce survival and dissemination of grampositive bacteria, including S. pyogenes, in several mouse models of disease [14–17]. Streptococcus pyogenes nuclease A (SpnA; Spy0747) is a chromosomally encoded, cellwall anchored DNase from S. pyogenes. Knocking out the gene encoding for SpnA led to lower survival of bacteria in human whole blood killing assays and attenuated virulence in a mouse model of infection [18, 19]. Conversely SpnA ‘gain of function’ mutants in avirulent Lactococcus lactis have been used to demonstrate that expression of SpnA promotes bacterial survival by reducing entrapment and destruction of bacteria by NETs [19]. SpnA/Spy0747 is highly conserved across strains and serotypes [18] and was recently identified as a possible vaccine candidate in a high-throughput screen for new GAS vaccine candidate antigens, emerging as the top-ranked candidate in the immunogenicity component of the screen with serum from 239 patients with pharyngitis [9]. Anti-SpnA antibodies are present in sera from patients recovering from invasive GAS disease, but are rarely detected in the early stages of GAS disease or in healthy individuals, suggesting this virulence factor is expressed during infection but does not elicit an enduring antibody response [19]. Therefore in this study we aimed to determine whether it was possible to stimulate a long-lived antibody response to SpnA, including the capacity to neutralise the DNase activity of this protein, by performing vaccination studies in mice. Vaccinated and control mice were then challenged with S. pyogenes to ascertain whether the presence of a preexisting antibody response to SpnA conferred any protection against development of disease.
Materials and methods Protein Mature recombinant SpnA protein was purified from an Escherichia coli BL21 strain with a pPROEX-Htb
13
Med Microbiol Immunol (2015) 204:185–191
expression vector (Life Technologies) containing spnA28–877 as described previously [19]. Cultures were grown at 30 °C and protein expression induced with 0.1 mM isopropyl-β-dthiogalactopyranoside (IPTG, Sigma). The (His)6-tagged proteins were purified on Ni+ 2 -NTA Sepharose (Sigma) as per the manufacturer’s instructions. SpnA was then dialysed overnight in 20 mM Tris pH 8.5, 5 mM CaCl2 and further purified by anion exchange over a HiLoad Q Sepharose column (GE Healthcare). rSpnA protein was eluted from the column with 1 M NaCl and the dominant peaks were concentrated then buffer exchanged into PBS. Protein purity was confirmed by 12.5 % SDS-PAGE and Coomassie blue staining. Vaccination Female CD1 mice were purchased from the Vernon Jansen Unit (VJU), University of Auckland, New Zealand. Animals were aged 5–6 weeks at the commencement of each study and were housed and cared for under SPF conditions in accordance with The Animal Welfare Act (1999) and institutional guidelines provided by the University of Auckland Animal Ethics Committee. A small number of mice (n = 4) were immunised intraperitoneally on days 0 and 14 with 20 μg rSpnA adsorbed to 1 mg aluminium hydroxide adjuvant (Alu–Gel–S, 2 % Al(OH)3, Serva Electrophoresis GmbH, Germany) per dose and serum collected on day 28 for use as a positive control for detection of anti-SpnA IgG responses by ELISA. For long-term vaccination studies protein antigen was diluted in sterile PBS, emulsified 1:1 in Incomplete Freund’s Adjuvant (IFA; Sigma) and delivered subcutaneously into the nape of the neck. Groups of 5–10 mice received a single vaccination with 50 μg rSpnA, or multiple vaccinations (weeks 0, 2, 4, 6) with 10 μg rSpnA. Age-matched mice were given an equivalent amount of IFA at these time points. Blood was sampled from the tail vein at regular intervals, collected in Microvette 500 serum-gel tubes (Sarstedt, Germany), processed and stored at −20 °C. S. pyogenes challenge S. pyogenes SF370 serotype M1 (ATCC 700294) was used as the challenge strain for these studies. An overnight broth culture of S. pyogenes was used to inoculate a 50 ml culture containing 10 ml bacteria and 40 ml fresh pre-warmed Brain Heart Infusion (BHI) broth (BD Biosciences) and grown until it reached an optical density A600 of 0.5. Bacteria were washed twice in PBS and re-suspended at an estimated concentration of 5 × 108 CFU/ml. The actual challenge dose was determined by plating serial tenfold dilutions of this suspension onto Columbia blood agar plates (Fort Richard, Auckland, New Zealand). Mice were anaesthetised with isoflurane and the flank area shaved, followed by subcutaneous administration
Med Microbiol Immunol (2015) 204:185–191
of 0.1 ml (~5 × 107 CFU) S. pyogenes. The animals were monitored daily for changes in condition and body weight. The lesions were measured daily using callipers and the lesion area calculated using the following formula (A = pi(L*W)/2) [20]. The experiments were terminated five (studies with multiple vaccinations) or eight days (single vaccination) after challenge. Mice were euthanised by CO2 inhalation, blood was collected by cardiac puncture to obtain post-challenge serum and lesion tissue excised. Lesion tissues were placed in sterile pre-weighed 2 ml collection tubes containing 2.8 mm ceramic beads (Omni International, USA) and 0.5 ml PBS. Tissue weights were recorded and tissues were homogenised with a Bead Ruptor 24 (Omni) for four cycles at 3.5 m/s for 10 s. Samples were serially diluted tenfold in PBS and each dilution spotted (25 μl per spot) in triplicate onto Columbia blood agar plates. Inoculated plates were dried at room temperature for several hours and incubated at 37 °C overnight. Colonies were counted and final data presented as CFU/lesion. Detection of anti‑SpnA IgG ELISAs were performed in Nunc maxisorb plates (Nunc A/S, Denmark). Wells were coated or incubated with 50 μl/ well protein, antibody or substrate. All incubation steps were for 1 h at room temperature and plates were washed thrice in PBS-0.05 % v/v Tween-20 between incubation steps. Wells were coated overnight at 4 °C with 1 μg/ml purified recombinant SpnA diluted in PBS. Wells were washed and then blocked with 200 μl/well PBS-1 % w/v bovine serum albumin (Life Technologies). Mouse serum was diluted 1/200 in blocking buffer and fivefold serial dilutions of serum then assayed in duplicate for each mouse per time point. Bound antibody was detected with goat anti-mouse IgG:HRP (Fcγ specific, Jackson ImmunoResearch) diluted 1:2,500. Peroxidase activity was detected using o-phenylenediamine dihydrochloride (OPD) substrate tablets (Zymed®, Life Technologies) diluted in 0.1 M citrate phosphate buffer as per the manufacturer’s instructions. The reaction was stopped with 10 % v/v HCl in water after 5 min. The absorbance was read at 490 nm on a μQuant Spectrophotometer (BioTek Instruments Inc.). Baseline and immune sera from mice immunised with rSpnA in Alum were used to establish the conditions for detection of anti-SpnA IgG and to produce a standard curve. A standard curve was made with a twofold dilution series of pooled day 28 immune sera in blocking buffer, starting from a dilution of 1:2,000. A standard curve was run on every plate to standardise the assay, a 4-parameter curve fit produced (KC-4 software package, Bio-Tek Instruments Inc.) and values interpolated from the linear part of the curve for all samples. IgG data were expressed as arbitrary ELISA Units, with a value of 1,000 corresponding to a 1:2,000 dilution of immune serum.
187
Serum neutralisation of SpnA Neutralisation of SpnA nuclease activity by human serum has previously been described [19], therefore serum from immunised mice was tested for inhibitory activity. Purified rSpnA was diluted in reaction buffer (40 mM Tris pH7.4, 20 mM CaCl2, 5 mM MgCl2) at a concentration of 0.375 μg/ml, mixed with an equivalent volume of undiluted mouse serum or dilution buffer (40 mM Tris pH 7.4) and kept on ice for 30 min. An equivalent volume of reaction buffer containing 5 μg salmon sperm DNA (Life Technologies) was then added and the mixture incubated at 37 °C for 1 h. The reaction was stopped by addition of loading dye and samples separated on a 1 % agarose gel. Every assay included positive and negative controls, including addition of neutralising serum from immunised mice; non-neutralising serum from naïve mice; and salmon sperm DNA added in the presence or absence of rSpnA. Each serum sample assessed for neutralising activity was scored as ‘+’ (neutralising) or ‘−’ (non-neutralising) on the basis of the presence or absence of salmon sperm DNA in the corresponding lane of the gel. Statistics All plots were created and statistical analyses performed using GraphPad Prism® 6.01 (GraphPad Software, Inc.), with p values of
