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. Author manuscript; available in PMC: 2016 Jan 15.
Published in final edited form as: Vaccine. 2014 Dec 6;33(4):523–526. doi: 10.1016/j.vaccine.2014.11.051

Protein A-neutralizing monoclonal antibody protects neonatal mice against Staphylococcus aureus

Vilasack Thammavongsa 1, Sabine Rauch 1, Hwan Keun Kim 1, Dominique M Missiakas 1, Olaf Schneewind 1,*
PMCID: PMC4378561  NIHMSID: NIHMS646789  PMID: 25488332

Abstract

Staphylococcus aureus is a cause of sepsis and meningitis in very-low-birth-weight (VLBW) infants. Clinical trials with S. aureus specific antibodies failed to protect VLBW neonates, which may be due to the immune evasive attributes of staphylococcal protein A (SpA). Here we show that mouse monoclonal antibody SpAKKAA-mAb 3F6, which neutralizes the immunoglobulin Fcγ-binding and B cell receptor crosslinking attributes of SpA, protects neonatal mice against S. aureus sepsis and raises protective immunity against subsequent staphylococcal infection. We developed a humanized SpAKKAA-mAb that protects neonatal mice against S. aureus sepsis and may therefore be subjected to clinical testing in VLBW neonates.

Keywords: Staphylococcus aureus, Staphylococcal protein A, Monoclonal antibody, Neonatal bacteremia and meningitis, Protective immunity, Recurrent infection

1. Introduction

Approximately 60,000 infants per year in the United States are very-low-birth-weight (VLBW), defined as <1,500 g and requiring hospitalization and intensive care [1]. The risk of late-onset (≥72 hours following birth) bacteremia or meningitis with Staphylococcus aureus is increased in VLBW neonates with decreasing birth weight and gestational age. In a recent study, 3.6% of VLBW infants were diagnosed with S. aureus bacteremia and/or meningitis, which caused 26% mortality [2]. Neonatal sepsis is also associated with severe neurodevelopmental and growth defects [3]. Due to their low gestational age, VLBW neonates lack maternal immunoglobulin, which is thought to increase susceptibility to staphylococcal infection [4]. Clinical trials have examined the efficacy of human antibodies against staphylococcal capsule (Altastaph®), clumping factor A (ClfA) and serine-aspartate repeat protein G (SdrG) (INH-A21) or monoclonal antibody against polyglycerol-phophate lipoteichoic acid (Pagibaximab®) to protect VLBW neonates against S. aureus infections [5–7]. Unfortunately, these trials failed to reach their study endpoints [8].

We hypothesize that the failure of antibodies to provide protection against S. aureus is based on the immune evasive attributes of staphylococcal protein A (SpA). Secreted SpA, which is either assembled in the envelope or released by bacteria, binds the Fcγ domain of immunoglobulins (Ig) as well as the Fab domains of VH3-type IgG and IgM [9, 10]. The Fcγ binding activity of SpA enables S. aureus to escape opsonophagocytic killing, whereas the crosslinking of VH3-type IgM B cell receptors disrupts the development of adaptive immune responses [11]. The non-toxigenic variant SpAKKAA is defective for immunoglobulin binding and, when used as immunogen, elicits SpA-neutralizing antibodies in mice and rabbits [12]. This enabled isolation of monoclonal antibodies, e.g. SpAKKAA-mAb 3F6, which protect adult mice against S. aureus disease and provide adjuvant function for the development of antibodies against many different staphylococcal antigens [13]. Here we examined the efficacy of mouse and humanized SpAKKAA-mAb to protect neonatal mice against S. aureus infection.

2. Materials and methods

2.1. Ethics statement

Experimental protocols were reviewed, approved and performed under supervision of The University of Chicago’s Institutional Biosafety Committee (IBC) and Institutional Animal Care and Use Committee (IACUC). FVB albino mice, used for their large litter size, were obtained from Charles River Laboratories. Mice received antibiotic-free water and food ad libitum and dams delivered approximately 10 pups following a 21–22 day gestation period.

2.2. Bacterial Strains

S. aureus USA300 LAC, a methicillin-resistant clinical isolate (MRSA), was grown in tryptic soy broth (TSB) at 37°C. Overnight cultures of S. aureus were diluted 1:100 into fresh TSB and grown for 3 hours at 37°C. Staphylococci were centrifuged, washed twice and diluted in PBS to A600 0.5 (2×108 CFU ml−1). Staphylococci were enumerated by colony formation on agar plates to quantify infectious doses.

2.3. Animal experiments

One-day-old pups were administered purified mAb SpAKKAA-3F6 or control antibody via intraperitoneal injection. Twenty-four hours later, pups were infected by subcutaneous injection cephalad to their tail with 1×103 CFU S. aureus USA300 LAC in 200 μl PBS. Pups were observed for survival and growth by weighing animals in daily intervals. Pups that survived the S. aureus challenge were weaned 21 days after birth; at 5 weeks of age, these mice were injected into the periorbital venous plexus with 5×106 CFU S. aureus USA300 LAC in 100 μl PBS and monitored for survival.

2.4. Enzyme linked immunosorbent assay

ELISA plates were coated with affinity purified SpAKKAA at 1 μg·ml−1 in 0.1 M carbonate buffer (pH 9.5) at 4°C overnight. Plates were blocked and incubated with dilutions of hyperimmune sera and developed using OptEIA reagent (BD Biosciences). For inhibition of non-immune binding of human IgG to protein A, purified SpA, SpAKK (Q9K and Q10K substitutions in each of the five IgBDs to abolish Fcγ binding) or SpAAA (D36A and D37A substitutions in each of the five IgBDs to abolish Fab′ binding) were used to coat ELISA plates [13]. Blocked plates were incubated with 50 μg·ml−1 human IgG1 monoclonal antibody control or humanized SpAKKAA-mAb prior to ligand binding. Plates were incubated with serial dilutions of human IgG conjugated to HRP and developed using OptEIA reagent. Half-maximal titers were calculated and normalized to human IgG1 control set at maximal binding.

2.5. Staphylococcal antigen matrix

Recombinant staphylococcal antigens were affinity purified - SpAKKAA, clumping factor A (ClfA) and B (ClfB), iron-regulated surface determinant B (IsdB), Panton-Valentin Leukocidin subunit F (LukF), coagulase (Coa) and von-Willebrand factor binding protein (vWbp) - and 2 μg each protein was blotted onto nitrocellulose membrane. Membranes were blocked with 5% whole milk, followed by incubation with hyper-immune sera (1:10,000 dilution). IRDye 680 conjugated affinity purified anti-mouse IgG (Rockland) was used to quantify signal intensities (A700) using the Odyssey™ infrared imaging system (Li-cor).

2.6. Humanized SpAKKAA-mAb

Humanized SpAKKAA-mAb was derived from the mouse monoclonal antibody SpAKKAA-mAb 3F6, which had been obtained by immunization of mice with SpAKKAA [12]. Complementary determining regions (CDR) of SpAKKAA-mAb 3F6 were grafted into a human IgG1 isotype antibody. The VH/VL sequence of SpAKKAA-mAb was cloned into an expression vector containing the genetic elements optimized for enhanced expression of antibodies in the dihydrofolate reductase-deficient mutant Chinese hamster ovary (DHFR-CHO) cell line DG44. Humanized SpAKKAA-mAb was affinity purified on protein A column and buffer exchanged into PBS.

3. Results

To develop a mouse model for S. aureus neonatal sepsis and efficacy testing of monoclonal antibodies, we bred FVB albino mice and obtained neonates that were infected via subcutaneous injection [14]. Forty-eight hours after birth, pups were assigned to specific experimental cohorts and injected with S. aureus USA300 LAC, the current epidemic MRSA strain in the United States. Initial experiments used infectious doses ranging from 1×102 to 1×106 CFU. All pups that were infected with a dose greater than ≥ 104 CFU ceased to gain weight and succumbed to S. aureus LAC challenge within 2–3 days (LD50 dose 1×103).

SpAKKAA-mAb 3F6 is an IgG2a mouse monoclonal antibody that binds each of the five immunoglobulin-binding domains of SpA (E, D, A, B and C) and neutralizes their association with mouse or human IgG and IgM [13]. SpAKKAA-mAb 3F6 promotes opsonophagocytic killing of S. aureus LAC in mouse blood [13]; administration of 5 mg·kg−1 of SpAKKAA-mAb 3F6 to adult BALB/c mice prevents S. aureus USA300 LAC abscess formation in renal tissues [13]. To examine SpAKKAA-mAb 3F6-mediated protection against neonatal sepsis in mice, 1-day-old pups received intraperitoneal injections with either SpAKKAA-mAb 3F6 or IgG2a (5 mg·kg−1 body weight) or were left untreated (mock)(Fig. 1A). Twenty-four hours later, on day-2 of life, pups were challenged by subcutaneous injection with 1×103 CFU S. aureus USA300 LAC. Sixty-percent of mock-treated pups succumbed to S. aureus challenge over the five day observation period (Fig. 1B). Survival and time-to-death were not affected by intraperitoneal injection of IgG2a control antibody (Fig. 1B). Eighty percent of neonates that received SpAKKAA-mAb 3F6 were protected against S. aureus USA300 LAC sepsis (mock vs. SpAKKAA-mAb 3F6, P=0.0374; IgG2a vs. SpAKKAA-mAb 3F6, P=0.0437). Pups that survived for 5 days did not show clinical signs of disease and developed similar to uninfected mice.

Fig. 1.

Fig. 1

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Vaccination with SpAKKAA-monoclonal antibody elicits protective immunity against S. aureus infection. (A) Experimental plan for the immunization of neonatal FVB albino mice with mouse SpAKKAA-mAb 3F6 (intraperitoneal injection of 5 mg kg−1), mouse IgG2a mAb (5 mg kg−1 isotype control) or mock treatment, which was followed 24 hours later by subcutaneous MRSA challenge with S. aureus USA300 LAC (B); survivors were bled on day 21 and serum analyzed for S. aureus specific antibodies (C) and challenged on day 35 by intravenous injection with S. aureus USA300 LAC (D). (B) Kaplan-Maier survival analysis of neonatal FVB albino mice infected via subcutaneous injection of 1×103 CFU S. aureus USA300 LAC to examine the efficacy of antibody treatment in preventing lethal sepsis in neonatal mouse cohorts (n=16 for each treatment group). Data shown are representative of 3 independent experiments. Statistical significance was determined with the Mantel-Cox test: mock vs. SpAKKAA-mAb 3F6, P=0.0374; IgG2a vs. SpAKKAA-mAb 3F6, P=0.0437. (C) Serum samples of previously infected mice were analyzed for antibodies against ClfA (clumping factor A), ClfB (clumping factor B), IsdB (iron surface determinant B), SpAKKAA, LukF (leukocidin F), Coa (coagulase), and vWbp (von Willebrand binding protein). IRDye 680 conjugated anti-mouse IgG was used to quantify signal intensities with the Odyssey™ infrared imaging system (Li-cor); brackets denote standard error of the means. (D) Kaplan-Meier survival analysis of 35 day old FVB albino mice that had been treated as described in (A&B) and received intravenous injection of 5×106 CFU S. aureus USA300 LAC. Data shown are representative of 3 independent experiments. Statistical significance was determined with the Mantel-Cox test: mock vs. naïve, P=0.1646; mock vs. SpAKKAA-mAb 3F6, P=0.0054; IgG2a vs. SpAKKAA-mAb 3F6, P=0.0072.

S. aureus infection in neonates, children or adults is not known to elicit protective immune responses and recurrent infection is a common feature of staphylococcal disease [11]. When examined in adult mice, S. aureus expression of SpA is required to block development of protective immune responses, which is attributable to its B cell superantigen activity [11]. We therefore examined whether neonatal mice that had survived S. aureus challenge were able to develop protective immune responses in the presence of SpAKKAA-mAb 3F6. Serum IgG levels against seven S. aureus antigens previously shown to contribute to vaccine protection in mice-ClfA [15], ClfB [16], IsdB [17], SpAKKAA [12] LukF [18], Coa and vWbp [19] were determined at 21 days post-challenge (Fig. 1C). As observed previously with adult BALB/c mice treated with SpAKKAA-mAb 3F6 [13], neonatal FVB mice that had been immunized with SpAKKAA-mAb 3F6 developed higher serum IgG titers against three S. aureus protective antigens: ClfA, IsdB and vWbp (Fig. 1C). In contrast to adult BALB/c mice immunized with SpAKKAA-mAb 3F6, the IgG level against IsdB was significantly increased in serum samples of SpAKKAA-mAb 3F6-immunized neonatal FVB mice (IgG2a vs. SpAKKAA-mAb 3F6, P<0.0001, two-way ANOVA) [13]. Of note, high levels of anti-SpAKKAA antibodies were detectable 21 days after passive transfer of SpAKKAA-mAb 3F6, which may be derived from newly produced anti-SpA antibodies or circulating SpAKKAA-mAb 3F6 (Fig. 1C). On day-35 of life, animals were challenged via intravenous injection with 5×106 CFU S. aureus USA300 LAC, a dose that causes lethal sepsis in adult FVB mice. IgG2a isotype control-treated mice as well as naïve, age-matched mice all succumbed to the intravenous S. aureus challenge within 2–3 days (Fig. 1D). However, cohorts of mice that had received SpAKKAA-mAb 3F6 on day-1 of life and had been infected 24 hours later displayed a significant delay in time-to-death and an increase in survival following challenge with an otherwise lethal dose of S. aureus USA300 LAC (IgG2a vs. SpAKKAA-mAb 3F6, P=0.0072).

We humanized SpAKKAA-mAb 3F6 and examined the half-life of circulating protein A specific antibody in neonatal mice. Following intraperitoneal injection of newborn mice with 10 mg·kg−1 antibody, animals (n=4) were bled at variable time intervals (days 1, 3, 7 and 14) and serum concentration of SpAKKAA-mAb 3F6 or humanized SpAKKAA-mAb was measured; this experiment revealed that humanized SpAKKAA-mAb has a shorter half-life in mice than SpAKKAA-mAb 3F6 (Fig. 2A). When examined for preclinical efficacy, intraperitoneal injection of humanized SpAKKAA-mAb protected neonatal mice against staphylococcal sepsis, induced by subcutaneous injection of 1×103 CFU S. aureus USA300 LAC, whereas the human IgG1 control antibody did not (human IgG1 vs. humanized SpAKKAA-mAb, P=0.0439) (Fig. 2B). Humanized SpAKKAA-mAb bound with high affinity to SpAKKAA (Ka 0.97 M ×1010 M−1) and inhibited the association of protein A (SpA) with human IgG (Fig. 2C). The neutralizing activity of the humanized SpAKKAA-mAb extends to both the Fcγ binding activity and the Fab VH3 crosslinking activity of protein A, as SpAKK (only binds Fab VH3) and SpAAA (only binds Fcγ) association with human immunoglobulin G (hIgG) was inhibited by humanized SpAKKAA-mAb but not by human IgG1 control (Fig. 2C).

Fig. 2.

Fig. 2

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Humanized SpAKKAA-mAb protects neonatal mice against S. aureus sepsis. (A) Following immunization via intraperitoneal injection with 10 mg kg−1 humanized SpAKKAA-mAb or 5 mg·kg−1 mouse monoclonal antibody SpAKKAA-mAb 3F6, neonatal FVB albino mice (n=4) were bled at timed intervals, serum concentration of antibodies determined by ELISA with SpAKKAA antigen; brackets denote standard error of the means. (B) Neonatal FVB albino mice on day-of-life 1 were immunized by intraperitoneal injection with humanized SpAKKAA-mAb (10 mg·kg−1), human IgG1 control antibody (10 mg·kg−1) or mock treated. Neonates were challenged by subcutaneous injection of 1×103 CFU S. aureus USA300 LAC on day-of-life 2. Kaplan-Maier survival analysis to examine the efficacy of antibody treatment in preventing lethal sepsis in neonatal mouse cohorts (n=10 for each treatment group). Data shown are representative of 2 independent experiments. Statistical significance was examined with the Mantel-Cox test: mock vs. hSpAKKAA-mAb, P=0.0114; hIgG1 vs. hSpAKKAA-mAb, P=0.0439. (C) Purified SpA, SpAKK or SpAAA were coated onto ELISA plates and subjected to binding experiments with human IgG (hIgG) in the presence of either humanized SpAKKAA-mAb (hSpAKKAA-mAb) or human IgG1 control antibody (hIgG1). Brackets denoted standard error of the means.

4. Discussion

VLBW neonates are at increased risk for bacterial sepsis and meningitis, which is associated with high mortality and long-term disability [1]. S. aureus is a major cause of neonatal sepsis and meningitis that, in spite of significant efforts, can currently not be addressed with a specific preventive strategy [2]. Previous failure of antibodies to achieve protection could be caused by SpA, an immunoglobulin-binding factor of S. aureus that disables humoral immune responses of infected hosts [11]. To test this hypothesis, we developed a neonatal mouse model for efficacy testing of monoclonal antibodies against S. aureus sepsis, which is based on earlier work examining antibiotic prophylaxis in neonatal mice [14]. Here, subcutaneous injection of a 200 μl suspension of ≥ 1×103 CFU S. aureus USA300 LAC caused lethal sepsis, defined by replication of staphylococci in tissues of infected hosts and rapid mortality. Compared to mock or isotype IgG2a control treated animals, SpAKKAA-mAb 3F6 protected neonatal mice against S. aureus sepsis. Moreover, the surviving mice that were treated with SpAKKAA-mAb 3F6 as pups elicited protective immune responses against subsequent challenge with an otherwise lethal intravenous dose of S. aureus USA300 LAC. These findings are in agreement with SpAKKAA-mAb 3F6 neutralization of SpA-dependent B cell superantigen activity during infection [13]. The SpAKKAA-mAb 3F6 antibody was humanized by grafting complementarity determining regions onto human IgG1. Humanized SpAKKAA-mAb 3F6 bound SpAKKAA with high affinity, blocked IgG binding to protein A, and provided protection against S. aureus sepsis in neonatal mice.

The molecular and cellular mechanisms promoting SpAKKAA-mAb 3F6 induced opsonophagocytosis of staphylococci in mice are not yet understood. For example, SpAKKAA-mAb 3F6 induced protection may involve complement deposition, complement receptor-mediated uptake of S. aureus and eventual killing of the pathogen by either neutrophils or macrophages. Alternatively, SpAKKAA-mAb 3F6 binding to protein A on the staphylococcal surface may promote Fcγ-receptor mediated uptake of the pathogen and killing by specific phagocyte populations [20]. The mouse model may provide important mechanistic insights into the establishment of protection against S. aureus that can subsequently be tested with human antibodies and human blood or phagocytic cells. To the best of our knowledge, data presented herein constitute the first demonstration that a monoclonal antibody can prevent S. aureus induced neonatal sepsis in animals and that it can function as an adjuvant to elicit immune responses protective against subsequent infection. Humanized SpAKKAA-mAb also protects neonatal mice against S. aureus sepsis. This antibody could therefore be tested in VLBW neonates to prevent S. aureus sepsis/meningitis. Nevertheless, efficacy studies for SpAKKAA-mAbs in the neonatal mouse model are currently limited to MRSA isolate LAC, a member of the non-capsulating USA300 clone epidemic in the United States [21]. Future work must examine SpAKKAA-mAb efficacy against other MRSA isolates including capsule expressing strains. Further, SpAKKAA-mAbs do not provide complete protection against S. aureus sepsis in mice, suggesting that additional antibodies may be needed to accomplish full protective immunity.

Highlights.

  • Neonatal mouse model for vaccine testing to prevent Staphylococcus aureus sepsis

  • Monoclonal antibody neutralizing protein A protects neonatal mice against sepsis

  • Development of a humanized monoclonal antibody neutralizing protein A

  • Humanized monoclonal antibody protects neonatal mice against sepsis

Acknowledgments

This work was supported by grants from the National Institute of Allergy and Infectious Diseases (AI052474, AI92711), the American Heart Association (POST4590023) and by the Innovation Fund of the University of Chicago Center for Technology Development & Ventures.

Footnotes

Disclosure of potential conflicts of interest

Vilasack Thammavongsa, Hwan Keun Kim, Dominique Missiakas and Olaf Schneewind declare a conflict of interest as inventors of patent applications that are related to the development of Staphylococcus aureus vaccines and are currently under commercial license.

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