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Immunotherapy targeting the Streptococcus pyogenes M protein or streptolysin O to treat or prevent influenza A superinfection

  • Andrea L. Herrera,

    Roles Data curation, Formal analysis, Investigation, Supervision, Writing – original draft

    Affiliation Division of Basic Biomedical Sciences, The Sanford School of Medicine of the University of South Dakota, Vermillion, SD, United States of America

  • Christopher Van Hove,

    Roles Data curation

    Affiliation Division of Basic Biomedical Sciences, The Sanford School of Medicine of the University of South Dakota, Vermillion, SD, United States of America

  • Mary Hanson,

    Roles Data curation

    Affiliation Division of Basic Biomedical Sciences, The Sanford School of Medicine of the University of South Dakota, Vermillion, SD, United States of America

  • James B. Dale,

    Roles Formal analysis, Funding acquisition, Resources, Writing – original draft, Writing – review & editing

    Affiliation Department of Medicine, Division of Infectious Diseases, University of Tennessee Health Science Center, Memphis, TN, United States of America

  • Rodney K. Tweten,

    Roles Resources, Writing – original draft, Writing – review & editing

    Affiliation Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, United States of America

  • Victor C. Huber,

    Roles Conceptualization, Methodology, Resources

    Affiliation Division of Basic Biomedical Sciences, The Sanford School of Medicine of the University of South Dakota, Vermillion, SD, United States of America

  • Diego Diel,

    Roles Funding acquisition, Resources

    Current address: Department of Population Medicine and Diagnostic Sciences, Animal Health Diagnostic Center, College of Veterinary Medicine, Cornell University, Ithaca, NY, United States of America

    Affiliation Department of Veterinary and Biomedical Sciences, South Dakota State University, Brookings, SD, United States of America

  • Michael S. Chaussee

    Roles Conceptualization, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Writing – original draft, Writing – review & editing

    michael.chaussee@usd.edu

    Affiliation Division of Basic Biomedical Sciences, The Sanford School of Medicine of the University of South Dakota, Vermillion, SD, United States of America

Immunotherapy targeting the Streptococcus pyogenes M protein or streptolysin O to treat or prevent influenza A superinfection

  • Andrea L. Herrera, 
  • Christopher Van Hove, 
  • Mary Hanson, 
  • James B. Dale, 
  • Rodney K. Tweten, 
  • Victor C. Huber, 
  • Diego Diel, 
  • Michael S. Chaussee
PLOS
x

Abstract

Viral infections complicated by a bacterial infection are typically referred to as coinfections or superinfections. Streptococcus pyogenes, the group A streptococcus (GAS), is not the most common bacteria associated with influenza A virus (IAV) superinfections but did cause significant mortality during the 2009 influenza pandemic even though all isolates are susceptible to penicillin. One approach to improve the outcome of these infections is to use passive immunization targeting GAS. To test this idea, we assessed the efficacy of passive immunotherapy using antisera against either the streptococcal M protein or streptolysin O (SLO) in a murine model of IAV-GAS superinfection. Prophylactic treatment of mice with antiserum to either SLO or the M protein decreased morbidity compared to mice treated with non-immune sera; however, neither significantly decreased mortality. Therapeutic use of antisera to SLO decreased morbidity compared to mice treated with non-immune sera but neither antisera significantly reduced mortality. Overall, the results suggest that further development of antibodies targeting the M protein or SLO may be a useful adjunct in the treatment of invasive GAS diseases, including IAV-GAS superinfections, which may be particularly important during influenza pandemics.

Introduction

Infection with influenza A virus (IAV) creates a permissive environment for secondary bacterial infections (often referred to as superinfections), which significantly increases the morbidity and mortality associated with both IAV epidemics and pandemics [1]. Streptococcus pyogenes (the group A Streptococcus, GAS) typically causes pharyngitis but also can cause more serious invasive GAS infections (iGAS) such as bacteremia, toxic shock syndrome, necrotizing fasciitis, and IAV superinfections. An analysis of lung biopsies from the 1918 influenza pandemic revealed that Streptococcus pneumoniae and GAS were the most frequently observed bacteria in the lungs and contributed to approximately 90% of the estimated 50 million deaths attributed to influenza [2].

IAV increases host susceptibility to secondary bacterial infections by a variety of mechanisms. Most research related to GAS has focused on the characterization of viral-induced changes affecting GAS adherence and internalization [37]. Consistent with these studies, murine models of IAV-GAS superinfection show that IAV infection significantly enhances the virulence of GAS [79]. In addition, active vaccination of mice against IAV provides significant protection against GAS secondary infections indicating a specific role for the virus in enhancing host susceptibility to superinfection [10, 11]. Finally, epidemiological studies support the idea that several viruses, and particularly influenza, increase the incidence of iGAS diseases including pneumonia [3, 12].

Many antibiotics, including penicillin, are effective against GAS. Nonetheless, iGAS diseases are associated with a surprisingly high fatality rate. For example, during the 2009 IAV pandemic, 7 out of 10 patients in California with a laboratory confirmed IAV-GAS superinfection died despite being treated with antibiotics and anti-viral agents. The median age at the time of death was 37 years [13]. In a separate study conducted between December 2010 and January 2011, 14 of 19 patients with an iGAS disease also had an IAV infection; ten died, even though at least nine received antibiotics effective against GAS ex vivo [14]. Finally, it is estimated that while only 12% of iGAS infections involve the lower respiratory tract [15], 38% are fatal [16]. Thus, while GAS remains susceptible to many antibiotics and influenza vaccines and anti-viral agents are widely used, the mortality of IAV-GAS superinfections is significant [3].

Currently there are no vaccines available for GAS; however, vaccination with the surface-localized M protein elicits protective opsonic antibodies [1720] and experimental M protein-based vaccines have been used in animal studies [21] and human clinical trials [22, 23]. In addition, we previously showed that active vaccination targeting the M protein confers 100% protection against mortality by using a murine model of IAV-GAS superinfection [24].

Another potential GAS vaccine target is the secreted cholesterol-dependent cytolysin (CDC) streptolysin O (SLO). SLO contributes to virulence [25] and orthologues are encoded in the genomes of a wide range of bacteria [26] including Streptococcus pneumoniae (pneumolysin; PLY), which has historically been the most frequent cause of IAV superinfections [27]. Passive immunotherapy with anti-PLY antibodies protects mice against S. pneumoniae bacteremia [28], indicating that the cytolytic CDC toxins may be good candidates for passive immunotherapy targeting bacterial pathogens associated with IAV superinfections.

While the development of an effective active vaccine against GAS is an ideal outcome, we were interested in assessing the efficacy of using passively administered antibodies to prevent or treat IAV-GAS superinfections. In the current study, we evaluated the prophylactic and therapeutic use of antisera targeting either the M protein or SLO in a murine model of IAV-GAS superinfection.

Materials and methods

Bacterial and viral isolates and culture conditions

S. pyogenes strain MGAS315 (serotype M3) was obtained from ATCC and grown statically with Todd-Hewitt broth, or agar plates (BD Biosciences, San Jose, CA) supplemented with 0.2% yeast extract (THY) at 37°C in 5% CO2. To prepare stocks to inoculate mice, GAS was grown overnight with THY agar, colonies were inoculated into pre-warmed THY medium, grown to the mid-exponential phase of growth (A600  =  0.5), and diluted in sterile phosphate-buffered saline (PBS; pH 7.4). The number of viable GAS was determined by dilution plating.

Influenza virus A/Puerto Rico/8/34-H1N1 (PR8), was propagated for 72 hours at 35°C in the allantoic cavities of 10-day-old embryonated chicken eggs [24, 29]. Viral RNA was extracted, reverse transcribed, and whole genome sequence analyses were used to confirm the genetic makeup of the viral preparation. Further characterization of the virus included calculating the median tissue culture infectious dose (TCID50) in Madin-Darby Canine Kidney (MDCK) cells.

Polyclonal antiserum

A recombinant hexavalent M protein vaccine containing protective M protein peptides from GAS serotypes M24, M5, M6, M19, M1, and M3 was generated by amplifying and ligating the 5’ end of each emm gene together into a pQE-30 vector [30]. The SLO toxoid was created by mutating amino acids (Thr and Leu) in the domain that comprises the cholesterol binding motif [31]. The hexavalent M protein or SLO toxoid were used to generate polyclonal antibodies in rabbits, as described previously [32]. Briefly, 1 mL of each purified recombinant protein (1.6 mg M protein or 2.5 mg SLO toxoid) was mixed 1:1 with the adjuvant Montanide ISA 50 (Seppic Inc; Fairfield, New Jersey). Adult 12-week old rabbits were kept in individual cages with food and water ad libitum throughout the study. Each rabbit was immunized with either antigen, which was administered by three 0.5 mL subcutaneous injections (day 0, 14, and 28) and one 0.5 mL intramuscular injection (day 0). The rabbits were euthanized by exsanguination 42 days after the initial injection.

Superinfection of mice and passive immunization

All experiments were conducted in conformity with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and according to the guidelines of the local Institutional Animal Care and Use Committee of the University of South Dakota (protocol number 10-06-18-21E). Drs. Victor Huber and Ruth Bakker (D.V.M.) provided all animal care and handling training. Female (6 to 8-week-old) BALB/c mice were purchased from Jackson Laboratories (Indianapolis, IN) and housed in groups of four with 24-hour access to food and water. The IAV-GAS superinfection model was previously described and we showed that IAV (PR8) persists in the lungs of mice up to day 9 after infection; which overlaps with GAS infection on day 7[33]. Mice were lightly anesthetized with 2.5% isoflurane and IAV or GAS was given intranasally (100 μl total volume). Intranasal administration was performed by pipetting small droplets every 2 to 3 seconds (approximately 5–10 μl) onto the outer edge of each nostril (alternating) where it was inhaled until 100 μl was delivered. For IAV and GAS the LD50 was determined by the method of Reed and Muench [34]. We used 0.1 LD50 as a sub-lethal dose, which was 100.75 TCID50 of IAV and 106 CFUs of GAS [11, 24]. For superinfections, mice were inoculated with IAV on day 0 and GAS on day 7. Animal health and behavior was monitored at least three times a day over the 21 day course of the experiment. Body weight was recorded daily. Endpoint criteria included extreme clinical signs of infection (huddling, hunched posture, ruffled fur, tachypnea), severe hypothermia as indicated by a temperature of 34°C (~4.5°C below normal), and weight loss equal to or greater than 20% of starting weight. Mice with one or more of these symptoms were immediately euthanized and the infection was considered lethal. The total number of mice used in the study was 138. Of these, 61 were euthanized and 77 succumbed to infection.

Polyclonal serum (125 μL) was administered either intraperitoneally (i.p.) using a 25-gauge needle, which was inserted at approximately 45° into the side of the abdominal wall, or administered intranasally (100 μL).

Antisera neutralization of SLO hemolytic activity

The capacity of SLO antisera to neutralize SLO cytolytic activity was determined as previously described [35]. Briefly, 100 μg of purified SLO toxin was serially diluted 2-fold with PBS (50 μl total volume) in a 96-well flat bottom plate. Then, 50 μl of SLO antiserum, non-immune serum, or PBS, was added and the plates were incubated at 37°C for 60 minutes. Next, 50 μl of 5% rabbit erythrocytes (Innovative Research; Novi, MI) was added to each well and incubated for an additional 30 minutes at 37°C. Intact erythrocytes were removed by centrifugation at 4,000 rpm for 5 minutes. Hemoglobin present in the supernatants was measured by determining the A540. The effective concentration of SLO required for 50% lysis (EC50) was determined with GraphPad Prism.

GAS quantification in mouse tissues

Mouse tissue samples were collected 24 hours after GAS inoculation (day 8). Blood was collected from the submandibular vein of each animal and immediately after they were euthanized by CO2 inhalation the lungs and spleens were removed and homogenized in sterile PBS. GAS were enumerated by dilution plating with THY or blood agar plates as previously described [11].

Enzyme-Linked Immunosorbent Assays (ELISA)

ELISA was done as previously described [21]. Briefly, 96-well plates were coated with 5 μg/mL of recombinant M protein or the SLO toxoid diluted with 0.1 M sodium carbonate (pH 9.8). Plates were blocked with 1% BSA, washed with PBS containing 0.05% Tween 20 (PBS-T), and serial dilutions of sera were added to the wells and incubated for 2 hours at 37°C. Plates were washed again with PBS-T, and HRP-conjugated goat anti-rabbit IgG (H+L) (Sigma, St. Louis, MO) was added to each well. After washing, HRP was detected using One-Step-TMB Turbo substrate (Thermo Scientific, Rockford, IL). The OD was measured at 450 nm using a Biotek EL808 plate reader (Biotek, Winooski, VT). End-point titers were defined as the reciprocal serum dilution corresponding with the last well demonstrating an OD450 of 0.1 in the titration curve. In some experiments, intact MGAS315 was immobilized to the ELISA plate as the antigen.

Bacterial killing in whole blood

Antibody-mediated killing of GAS was measured with whole mouse blood (Biochemed Services; Winchester VA). Stocks of GAS were diluted in PBS to 104 CFUs and mixed with 100 μg of IgG (diluted in PBS) and incubated at room temperature for 15 minutes to allow antibodies to bind GAS. Then, 200 μL of whole mouse blood was added and incubated for 30 minutes at 37°C and 5% CO2. Viable GAS were enumerated by dilution plating on THY agar. Cytochalasin D inhibits actin polymerization, and is a general inhibitor of phagocytosis. In some experiments cytochalasin D (final concentration, 10 μM; Sigma-Aldrich, St. Louis, MO) was added to whole blood 30 minutes prior to the addition of GAS to inhibit phagocytosis.

IgG purification

The IgG fraction of antibodies was isolated from polyclonal rabbit sera using a 1 mL Melon Gel column, as per manufacturer’s instructions (Thermo Fisher Scientific; Rockford, IL). All purified IgG was immediately buffer exchanged to a final concentration of 1 mg/mL in PBS using 40 kDa Zeba spin columns (Thermo Fisher Scientific). IgG fractionation was verified by SDS-PAGE and concentrations were determined by absorbance at 280 nm using an extinction coefficient (E280 0.1%) of 1.4.

Statistical analysis

Data were analyzed using GraphPad Prism software (GraphPad Software, Inc., La Jolla, CA), and values were accepted as significant if P < 0.05. Statistical analyses included a one-way or two-way analysis of variance (ANOVA) with a Tukey’s multiple comparisons post-hoc test, Kaplan Meier survival analysis, or a Student’s t-test, as appropriate. All figures were created using GraphPad Prism software.

Results

Characterization of antisera to SLO and the M protein

Rabbits were injected with either a recombinant hexavalent M-protein vaccine, which included an amino acid sequence known to elicit antibodies specific to the serotype M3 protein [30] or with an SLO toxoid [26]. The titers of antigen-specific antibodies against the SLO or the M protein vaccines were determined by using an enzyme-linked immunosorbent assay (ELISA) and were 600,000 and 400,000 respectively (Fig 1). Antisera to SLO and to the M protein also contained antibodies that bound to an immobilized serotype M3 strain MGAS315 (antibody titers of 22,000 and 6,000 respectively), (Fig 1). The results indicated that the epitopes of the target proteins were exposed and accessible to the antibodies. Non-immune serum, which in subsequent experiments was used as a negative control, also contained a low level of antibodies that bound to immobilized MGAS315 (antibody titer 1,000).

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Fig 1. Antisera against SLO or the M protein bound to purified antigen and to immobilized GAS strain MGAS315.

(A). The titers were determined with ELISA. Data shown are the means ± sem from 4 to 5 independent experiments. n.d. indicates antibody binding was not detected; α refers to antisera. ELISA was also used to determine the titers of antibodies specific to SLO (open square) or the M protein (open diamond) in the blood and lungs of mice 24 hours after B) i.p. or C) i.n administration of antisera. The means ± sem from 3 to 9 mice are indicated. Statistical significance was determined with the Student's t-test.

https://doi.org/10.1371/journal.pone.0235139.g001

Determination of antibody titers to SLO and the M protein in the blood and lungs of mice following intraperitoneal (i.p.) or intranasal (i.n.) administration of antisera

Prior to assessing the efficacy of using passively administered antisera to curtail disease, it was of interest to measure the titers of antibodies in mice following either i.p. or i.n. administration of antisera. Antisera to either SLO or the M protein was given separately by either i.p. (125 μL) or i.n. (100 μL) inoculation. After 24 h, the titers of antigen-specific antibodies in the blood and lungs of mice were determined with ELISA (Fig 1B and 1C). Following i.p. administration, the antibody titers to SLO (50,000) and the M protein (20,000) were similar in the blood (p > 0.05); however, the average antibody titer to SLO (5,000) was greater in the lungs compared to the titer of M protein-specific antibodies (<1,000; p < 0.05; Fig 1B). There was significantly greater variation in antibody titers among lung samples compared to blood samples following i.p. injection (SLO F8,5 = 95, p < 0.0005 and M protein F8,5 = 6560, p < 0.0005; Fig 1B). Following i.n. administration of antisera, the antibody titers to SLO and the M protein in both the blood and lungs were similar and below 1,000 (Fig 1C).

Growth inhibition of GAS in murine blood in the presence of antibodies to the M protein and antibody-mediated neutralization of SLO cytolytic activity

We next determined if the IgG fraction of the antisera to the M protein could inhibit the growth of GAS in the presence of phagocytes and complement. For this, GAS was incubated for 30 minutes with the IgG fraction of antisera raised against the M protein; controls included non-immune rabbit IgG. The bacteria were then suspended with murine blood for 30 minutes at 37°C and viable bacteria were enumerated by dilution plating. As an additional control, cytochalasin D was used to inhibit actin polymerization and phagocytosis. Compared to non-immune IgG, the addition of antibodies to the M protein decreased the number of viable GAS by 90%; however, the difference was not statistically significant (Fig 2). The addition of cytochalasin D inhibited the killing of GAS incubated with antibodies to the M protein (p < 0.05; Fig 2); however, the addition of cytochalasin D did not inhibit the killing of GAS incubated with non-immune IgG. The results suggested the antibodies to the M protein enhanced phagocytosis and killing of GAS in murine blood.

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Fig 2. Antibodies to the M protein inhibit growth of GAS in blood.

Growth and viability of GAS in murine blood. GAS was pre-incubated for 30 min. with IgG to the M protein or non-immune IgG (ctrl) prior to suspension in murine blood that contained, or not, cytochalasin D. After an additional 30 min., viable bacteria were enumerated by dilution plating. The results shown are the means ± sem from 4 independent experiments. Statistical significance was determined with the Student's t-test.

https://doi.org/10.1371/journal.pone.0235139.g002

To determine if the antisera to SLO contained antibodies that neutralized SLO cytolytic activity, we measured SLO-mediated hemolysis in the presence of antiserum to SLO, non-immune serum, or PBS. The SLO-specific antisera inhibited hemolytic activity compared to controls (p < 0.0001; Table 1) indicating the sera contained neutralizing antibodies.

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Table 1. Hemolytic activity of SLO in the presence of serum.

https://doi.org/10.1371/journal.pone.0235139.t001

Determination of the abundance of GAS in the lungs, spleens, and blood of IAV-GAS superinfected mice treated prophylactically with antisera to SLO or the M protein

To determine if prophylactic immunotherapy can decrease the bacterial burden among IAV-GAS superinfected mice, we quantified GAS in the lungs, blood, and spleen of superinfected mice. To do so, mice were infected i.n. with a sublethal dose of IAV (0.1 LD50). Seven days later they were given antisera to either SLO or the M protein, or non-immune serum. Six hours later, mice were inoculated i.n. with a sublethal dose of GAS (0.1 LD50). After 24 h, the mice were euthanized and the number of viable bacteria in the lungs, blood, and spleen was determined by dilution plating. The differences among individual groups treated with antisera compared to controls (e.g. antisera to M protein versus control sera) were not statistically significant (p > 0.05; Fig 3); however, when results were analyzed by comparing the combination of mice treated with either antisera, there were fewer bacteria in the lungs and blood (p < 0.05) of mice compared to mice treated with non-immune sera.

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Fig 3. Quantification of GAS in the lungs, blood, and spleens of mice treated prophylactically with antisera.

Mice were infected i.n. with 0.1 LD50 IAV on day 0. On day 7, mice were treated with antisera specific to either the M protein or SLO, or non-immune sera (ctrl) and superinfected 6 h later with GAS (0.1 LD50). Mice were euthanized 24 h later and the number of GAS present in blood, lungs, and spleens was determined by dilution plating. Differences between the individual groups were not statistically significant (p > 0.05); however, the difference in the number of bacteria in the lungs and blood of mice treated with either antisera to SLO or the M protein was lower compared to those treated with non-immune sera (p < 0.05; *). Statistical significance was determined with the Student's t-test.

https://doi.org/10.1371/journal.pone.0235139.g003

Determination of the morbidity and mortality of superinfected mice treated prophylactically with antisera to SLO or the M protein

To determine if prophylactic use of antisera to SLO or the M protein could decrease either the morbidity or mortality associated with IAV-GAS superinfection, groups of mice were first inoculated with a sublethal dose of IAV (0.1 LD50) on day 0. On day 7 mice were given antisera to either SLO or the M protein, or non-immune rabbit sera. Six hours later, mice were inoculated with a sublethal dose of GAS (0.1 LD50). Additional untreated groups of mice included those infected with a sublethal dose (0.1 LD50) of either IAV or GAS alone to control for the synergistic effects of viral-bacterial superinfection. Prophylaxis with antisera specific to SLO and to the M protein significantly decreased morbidity compared to mice receiving non-immune sera (Fig 4A; p < 0.001). Prophylaxis with antisera to either SLO or the M protein also increased survival by 37.5% and 12.5%, respectively compared to mice receiving non-immune sera (p > 0.05; Fig 4B).

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Fig 4. Determination of the effect of prophylactic administration of antisera to SLO (α-SLO) or the M protein (α-M) on morbidity (weight loss) and mortality of IAV-GAS superinfected mice.

A) Treatment with α-SLO and α-M decreased morbidity compared to groups of mice treated with non-immune sera (p < 0.001) B) The mortality associated with IAV-GAS superinfection was slightly reduced following administration of either α-SLO or α-M compared to treatment with non-immune sera (p > 0.05). Control groups also included untreated mice inoculated with IAV alone (day 0) or GAS alone (day 7). The area under the curve analysis (days 8–15) was completed prior to an unpaired Student’s t-test to compare the differences in morbidity. A Kaplan–Meier survival analysis was used to compare the differences in mortality.

https://doi.org/10.1371/journal.pone.0235139.g004

Determination of the abundance of GAS in the lungs, spleens, and blood of IAV-GAS superinfected mice treated with antisera to SLO or the M protein

To determine if immunotherapy administered after the establishment of an IAV-GAS superinfection could enhance the clearance of GAS from mice, we quantified GAS in the lungs, blood, and spleen of superinfected mice treated with antisera to either SLO or the M protein. The differences between each individual group treated with immune sera compared to mice treated with non-immune sera (e.g. antisera to M protein versus control sera) were not statistically significant (p > 0.05; Fig 5); however, when we compared mice treated with antisera to either SLO or the M protein (as a combined group) the mice treated with GAS-specific antisera had fewer GAS in the lungs and spleens compared to mice treated with non-immune sera (p < 0.05; Fig 5).

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Fig 5. Quantification of GAS in the lungs, blood, and spleens of mice treated with antisera 2 h after superinfection.

Mice were infected i.n. with 0.1 LD50 IAV on day 0 and GAS on day 7 followed 2 h later by treatment with antisera to the M protein, SLO, or non-immune sera as a control (ctrl). Mice were euthanized 24 h later and the number of GAS present in blood, lungs, and spleens was determined by dilution plating. Differences between the individual groups were not statistically significant (p > 0.05); however, there were fewer GAS in the lungs and spleen of mice treated with either antisera to SLO or the M protein compared to those treated with non-immune sera (p < 0.05; *). Statistical significance was determined with the Student's t-test.

https://doi.org/10.1371/journal.pone.0235139.g005

Determination of the morbidity and mortality of superinfected mice treated with antisera to SLO or the M protein

To determine if treatment with antisera to SLO or the M protein could decrease the morbidity or mortality associated with an established IAV-GAS superinfection, mice were first inoculated with a sublethal dose of IAV (0.1 LD50) on day 0. On day 7 mice were inoculated with a sublethal dose of GAS (0.1 LD50) and 2 h later were given antisera to either SLO or the M protein. As a control, mice were similarly treated with non-immune sera. Treatment with antisera to the M protein did not decrease morbidity compared to mice receiving non-immune sera (p > 0.05; Fig 6A). Treatment with antisera to SLO decreased morbidity compared to mice treated with non-immune sera (p < 0.001; Fig 6A); however, treatment with neither antisera significantly reduced mortality compared mice treated with non-immune sera (p > 0.05; Fig 6B).

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Fig 6. Determination of the effect of therapeutic administration of antisera to SLO (α-SLO) or the M protein (α-M) on morbidity (weight loss) and mortality of IAV-GAS superinfected mice.

Mice were injected with antibodies specific to SLO (α-SLO) or the M protein (α-M) 2 h after superinfection with GAS. For controls, groups of mice were treated with non-immune sera (α-ctrl). Treatment with α-SLO decreased morbidity compared to treatment with non-immune sera (p < 0.001). There were no differences in mortality between treated and untreated groups of mice (p > 0.05). The area under the curve analysis (days 8–15) was completed prior to an unpaired Student’s t-test to compare the differences in morbidity. A Kaplan–Meier survival analysis was used to compare the differences in mortality between the groups.

https://doi.org/10.1371/journal.pone.0235139.g006

Discussion

Despite the availability of influenza vaccines, anti-viral agents, and antibiotics that are effective against GAS ex vivo, the morbidity and mortality of IAV-GAS superinfections is considerable. We assessed the use of passive immunotherapy to reduce morbidity and mortality using an established murine model of IAV-GAS superinfection and antisera targeting two GAS proteins; one against the cell wall-associated M protein and the other against the exoprotein SLO. We measured efficacy with three endpoints: i) enumeration of GAS in the blood and organs of mice. ii) weight loss (morbidity). iii) mortality. In general, prophylactic use of the antisera decreased morbidity compared to groups of mice treated with non-immune sera. Therapeutic treatment with the antisera slightly decreased the numbers of bacteria in the lungs and spleens of superinfected mice but there was no significant decrease in mortality. While refinement of the antibody formulation is necessary, our results support the continued development of passive immunotherapy as a potential preventative measure, or treatment, for IAV-GAS superinfections.

Both the M protein and SLO enhance virulence by decreasing the susceptibility of GAS to killing by immune effector cells such as macrophages and neutrophils [3640]. Passive antibody treatment can decrease disease by two or more mechanisms. First, antibodies can promote opsonophagocytosis, which is consistent with our interpretation of results assessing the survival of GAS incubated with antibodies to the M protein and suspended in whole blood (Fig 2). In this regard, serotype-specific opsonic antibodies to the cell wall localized M protein have been known for decades to be important in the clearance of GAS and are typically protective [41]. In contrast, SLO is an exoprotein that disrupts the integrity of cholesterol-containing cell membranes [36], which can induce caspase-dependent apoptosis in neutrophils, macrophages [37], and epithelial cells [38]. Antibody-mediated neutralization of SLO [42] can decrease toxin-mediated apoptosis of immune effector cells and thereby increase the killing of GAS. Similarly, antibodies to the exoprotein streptolysin S enhanced GAS killing by neutralizing the cytolytic activity of the toxin [43]. Finally, active vaccination of non-human primates with an SLO toxoid (also containing other GAS antigens) induced non-opsonizing SLO-specific antibodies and decreased pharyngitis [44]. Thus, the antisera to the M protein and SLO likely enhanced opsonophagocytosis (Fig 2) and neutralized cytolytic activity (Table 1), respectively.

Our study was not designed to compare the efficacy of antisera against the M protein to that against SLO but rather to assess the feasibility of pursuing such an approach. We note that treatment was not based on the titer of antigen-specific antibodies, which differed between the sera.

The mortality of iGAS diseases is remarkably high despite the susceptibility of the pathogen to antibiotics and there is considerable need for adjunct therapeutics. Patients with iGAS diseases typically have either low titers, or lack antibodies to the causative GAS serotype suggesting that sufficient levels of circulating antibodies may protect against iGAS diseases [45, 46]. Intravenous immunoglobulin (IVIG) contains both opsonic and neutralizing antibodies to several GAS virulence factors [4752]. There has been some clinical success using IVIG to treat iGAS diseases, especially streptococcal toxic shock syndrome [51, 53, 54]; however, definitive results from robust clinical trials are lacking [55, 56]. This is due, in part, to challenges in enrolling a sufficient number of patients and probably the low concentration, and variability, of GAS-specific antibodies in IVIG [57]. A study supporting this interpretation found that when IgG antibodies specific to GAS were purified from human IVIG they were more efficacious when used as a passive vaccine in a murine model of iGAS infection compared to IVIG [58]. These results support the idea that GAS-specific antibodies may be an effective therapeutic approach to manage iGAS diseases, although it is acknowledged that IVIG treatment also dampens the inflammatory response [59], which likely affects clinical outcomes.

Passive antibody therapy has been successfully used to treat toxin-mediated bacterial diseases including diphtheria, tetanus, botulism, and Clostridium difficile infections [60]. These diseases result largely from the activity of one, or two, specific proteins. In contrast, the pathogenesis of GAS (and many other bacteria), involves multiple, sometimes functionally redundant, virulence factors. Thus, the successful development of antibody-based adjunct therapeutics to pathogens employing multiple virulence factors may require formulations that include antibodies to multiple virulence factors and not monoclonal or polyclonal antibodies targeting a single protein. Subsequent studies will explore the efficacy of using a combination of IgG antibodies to both the M protein and SLO in mitigating the morbidity and mortality of IAV-GAS superinfections.

Several studies have used animal models to test the ability of passive immunotherapy to protect against iGAS diseases, although ours is the first to assess efficacy in the context of a viral superinfection. Previous studies showed that opsonic antisera to a fibronectin binding protein (FBP54) [61], the streptococcal hemoprotein receptor (Shr) [62], GAS carbohydrate [63], or various epitopes of the M protein [6466] are protective compared to controls. In addition, passive immunization with non-opsonic antisera containing antibodies to Streptococcal esterase (Sse) [67], streptococcal pyrogenic exotoxin A (SpeA) [68], the M6 protein [69], or C5a peptidase [70] are also protective based on studies using mice. Taken together, the studies show that the prophylactic use of either opsonizing or non-opsonizing antibodies targeting GAS can be useful in the protecting against a GAS monoinfection; however, there has been less success in treating established iGAS infections using passive immunotherapy.

While an active vaccine against GAS is an ideal outcome, many successful vaccines do not abolish disease including the pneumococcal, Hib, and influenza vaccines [7174]. Therefore, the development of therapeutic antibodies designed as an adjunct treatment for severe iGAS diseases, including IAV-GAS superinfections, may prove to be beneficial, particularly during influenza pandemics.

References

  1. 1. Morens DM, Taubenberger JK, Fauci AS. Predominant role of bacterial pneumonia as a cause of death in pandemic influenza: implications for pandemic influenza preparedness. The Journal of infectious diseases. 2008;198(7):962–70. Epub 2008/08/20. pmid:18710327; PubMed Central PMCID: PMC2599911.
  2. 2. Morens DM, Fauci AS. The 1918 influenza pandemic: insights for the 21st century. The Journal of infectious diseases. 2007;195(7):1018–28. Epub 2007/03/03. pmid:17330793.
  3. 3. Herrera AL, Huber VC, Chaussee MS. The Association between Invasive Group A Streptococcal Diseases and Viral Respiratory Tract Infections. Frontiers in microbiology. 2016;7:342. Epub 2016/04/06. pmid:27047460; PubMed Central PMCID: PMC4800185.
  4. 4. Herrera AL, Suso K, Allison S, Simon A, Schlenker E, Huber VC, et al. Binding host proteins to the M protein contributes to the mortality associated with influenza-Streptococcus pyogenes superinfections. Microbiology (Reading, England). 2017. Epub 2017/09/26. pmid:28942759.
  5. 5. Herrera AL, Faal H, Moss D, Addengast L, Fanta L, Eyster K, et al. The Streptococcus pyogenes fibronectin/tenascin-binding protein PrtF.2 contributes to virulence in an influenza superinfection. Scientific reports. 2018;8(1):12126. Epub 2018/08/16. pmid:30108238; PubMed Central PMCID: PMC6092322.
  6. 6. Okamoto S, Nagase S. Pathogenic mechanisms of invasive group A Streptococcus infections by influenza virus-group A Streptococcus superinfection. Microbiology and immunology. 2018;62(3):141–9. Epub 2018/01/30. pmid:29377225.
  7. 7. Okamoto S, Kawabata S, Terao Y, Fujitaka H, Okuno Y, Hamada S. The Streptococcus pyogenes capsule is required for adhesion of bacteria to virus-infected alveolar epithelial cells and lethal bacterial-viral superinfection. Infection and immunity. 2004;72(10):6068–75. Epub 2004/09/24. pmid:15385511; PubMed Central PMCID: PMC517596.
  8. 8. Okamoto S, Kawabata S, Nakagawa I, Okuno Y, Goto T, Sano K, et al. Influenza A virus-infected hosts boost an invasive type of Streptococcus pyogenes infection in mice. Journal of virology. 2003;77(7):4104–12. Epub 2003/03/14. pmid:12634369; PubMed Central PMCID: PMC150641.
  9. 9. Weeks-Gorospe JN, Hurtig HR, Iverson AR, Schuneman MJ, Webby RJ, McCullers JA, et al. Naturally occurring swine influenza A virus PB1-F2 phenotypes that contribute to superinfection with Gram-positive respiratory pathogens. Journal of virology. 2012;86(17):9035–43. Epub 2012/06/08. pmid:22674997; PubMed Central PMCID: PMC3416121.
  10. 10. Okamoto S, Kawabata S, Fujitaka H, Uehira T, Okuno Y, Hamada S. Vaccination with formalin-inactivated influenza vaccine protects mice against lethal influenza Streptococcus pyogenes superinfection. Vaccine. 2004;22(21–22):2887–93. Epub 2004/07/13. pmid:15246625.
  11. 11. Chaussee MS, Sandbulte HR, Schuneman MJ, Depaula FP, Addengast LA, Schlenker EH, et al. Inactivated and live, attenuated influenza vaccines protect mice against influenza: Streptococcus pyogenes super-infections. Vaccine. 2011;29(21):3773–81. Epub 2011/03/29. pmid:21440037; PubMed Central PMCID: PMC3084433.
  12. 12. de Gier B VB, Woudt SHS, van Sorge NM, van Asten L. Associations between common respiratory viruses and invasive group A streptococcal infection: A time-series analysis. Influenza and other respiratory viruses. 2019;2019 Sep;13(5):453–458. Epub 2019 Jun 25. pmid:31237087
  13. 13. Jean C, Louie JK, Glaser CA, Harriman K, Hacker JK, Aranki F, et al. Invasive group A streptococcal infection concurrent with 2009 H1N1 influenza. Clinical infectious diseases: an official publication of the Infectious Diseases Society of America. 2010;50(10):e59–62. Epub 2010/04/10. pmid:20377405.
  14. 14. Scaber J, Saeed S, Ihekweazu C, Efstratiou A, McCarthy N, O'Moore E. Group A streptococcal infections during the seasonal influenza outbreak 2010/11 in South East England. Euro surveillance: bulletin Europeen sur les maladies transmissibles = European communicable disease bulletin. 2011;16(5). Epub 2011/02/15. pmid:21315058.
  15. 15. Olsen RJ, Ashraf M, Gonulal VE, Ayeras AA, Cantu C, Shea PR, et al. Lower respiratory tract infection in cynomolgus macaques (Macaca fascicularis) infected with group A Streptococcus. Microbial pathogenesis. 2010;49(6):336–47. Epub 2010/08/03. pmid:20674736.
  16. 16. Muller MP, Low DE, Green KA, Simor AE, Loeb M, Gregson D, et al. Clinical and epidemiologic features of group a streptococcal pneumonia in Ontario, Canada. Archives of internal medicine. 2003;163(4):467–72. Epub 2003/02/18. pmid:12588207.
  17. 17. Batzloff MR, Hayman WA, Davies MR, Zeng M, Pruksakorn S, Brandt ER, et al. Protection against group A streptococcus by immunization with J8-diphtheria toxoid: contribution of J8- and diphtheria toxoid-specific antibodies to protection. The Journal of infectious diseases. 2003;187(10):1598–608. Epub 2003/05/02. pmid:12721940.
  18. 18. Brandt ER, Teh T, Relf WA, Hobb RI, Good MF. Protective and nonprotective epitopes from amino termini of M proteins from Australian aboriginal isolates and reference strains of group A streptococci. Infection and immunity. 2000;68(12):6587–94. Epub 2000/11/18. pmid:11083769; PubMed Central PMCID: PMC97754.
  19. 19. Bronze MS, Courtney HS, Dale JB. Epitopes of group A streptococcal M protein that evoke cross-protective local immune responses. Journal of immunology (Baltimore, Md: 1950). 1992;148(3):888–93. Epub 1992/02/01. pmid:1370521.
  20. 20. Olive C, Batzloff MR, Horvath A, Wong A, Clair T, Yarwood P, et al. A lipid core peptide construct containing a conserved region determinant of the group A streptococcal M protein elicits heterologous opsonic antibodies. Infection and immunity. 2002;70(5):2734–8. Epub 2002/04/16. pmid:11953422; PubMed Central PMCID: PMC127950.
  21. 21. Hall MA, Stroop SD, Hu MC, Walls MA, Reddish MA, Burt DS, et al. Intranasal immunization with multivalent group A streptococcal vaccines protects mice against intranasal challenge infections. Infection and immunity. 2004;72(5):2507–12. Epub 2004/04/23. pmid:15102757; PubMed Central PMCID: PMC387888.
  22. 22. Kotloff KL, Corretti M, Palmer K, Campbell JD, Reddish MA, Hu MC, et al. Safety and immunogenicity of a recombinant multivalent group a streptococcal vaccine in healthy adults: phase 1 trial. Jama. 2004;292(6):709–15. Epub 2004/08/12. pmid:15304468.
  23. 23. McNeil SA, Halperin SA, Langley JM, Smith B, Warren A, Sharratt GP, et al. Safety and immunogenicity of 26-valent group a streptococcus vaccine in healthy adult volunteers. Clinical infectious diseases: an official publication of the Infectious Diseases Society of America. 2005;41(8):1114–22. Epub 2005/09/16. pmid:16163629.
  24. 24. Klonoski JM, Hurtig HR, Juber BA, Schuneman MJ, Bickett TE, Svendsen JM, et al. Vaccination against the M protein of Streptococcus pyogenes prevents death after influenza virus: S. pyogenes super-infection. Vaccine. 2014;32(40):5241–9. Epub 2014/08/01. pmid:25077423; PubMed Central PMCID: PMC4146501.
  25. 25. Limbago B, Penumalli V, Weinrick B, Scott JR. Role of streptolysin O in a mouse model of invasive group A streptococcal disease. Infection and immunity. 2000;68(11):6384–90. Epub 2000/10/18. pmid:11035749; PubMed Central PMCID: PMC97723.
  26. 26. Tweten RK, Hotze EM, Wade KR. The Unique Molecular Choreography of Giant Pore Formation by the Cholesterol-Dependent Cytolysins of Gram-Positive Bacteria. Annual review of microbiology. 2015;69:323–40. Epub 2015/10/22. pmid:26488276.
  27. 27. McCullers JA. Insights into the interaction between influenza virus and pneumococcus. Clinical microbiology reviews. 2006;19(3):571–82. Epub 2006/07/19. pmid:16847087; PubMed Central PMCID: PMC1539103.
  28. 28. Saeland E, Vidarsson G, Leusen JH, Van Garderen E, Nahm MH, Vile-Weekhout H, et al. Central role of complement in passive protection by human IgG1 and IgG2 anti-pneumococcal antibodies in mice. Journal of immunology (Baltimore, Md: 1950). 2003;170(12):6158–64. Epub 2003/06/10. pmid:12794146.
  29. 29. Huber VC, Thomas PG, McCullers JA. A multi-valent vaccine approach that elicits broad immunity within an influenza subtype. Vaccine. 2009;27(8):1192–200. Epub 2009/01/13. pmid:19135117; PubMed Central PMCID: PMC2663794.
  30. 30. Dale JB. Multivalent group A streptococcal vaccine designed to optimize the immunogenicity of six tandem M protein fragments. Vaccine. 1999;17(2):193–200. Epub 1999/02/13. pmid:9987154.
  31. 31. Farrand S, Hotze E, Friese P, Hollingshead SK, Smith DF, Cummings RD, et al. Characterization of a streptococcal cholesterol-dependent cytolysin with a lewis y and b specific lectin domain. Biochemistry. 2008;47(27):7097–107. Epub 2008/06/17. pmid:18553932; PubMed Central PMCID: PMC2622431.
  32. 32. Fernandes MHV, Maggioli MF, Otta J, Joshi LR, Lawson S, Diel DG. Senecavirus A 3C Protease Mediates Host Cell Apoptosis Late in Infection. Frontiers in immunology. 2019;10:363. Epub 2019/03/29. pmid:30918505; PubMed Central PMCID: PMC6424860.
  33. 33. Klonoski JM, Watson T, Bickett TE, Svendsen JM, Gau TJ, Britt A, et al. Contributions of Influenza Virus Hemagglutinin and Host Immune Responses Toward the Severity of Influenza Virus: Streptococcus pyogenes Superinfections. Viral immunology. 2018;31(6):457–69. Epub 2018/06/06. pmid:29870311; PubMed Central PMCID: PMC6043403.
  34. 34. Reed LJ. MH. A simple method of estimating fifty per cent endpoints. Am J Hyg. 1938;27:493–7.
  35. 35. Hotze EM, Le HM, Sieber JR, Bruxvoort C, McInerney MJ, Tweten RK. Identification and characterization of the first cholesterol-dependent cytolysins from Gram-negative bacteria. Infection and immunity. 2013;81(1):216–25. Epub 2012/11/02. pmid:23115036; PubMed Central PMCID: PMC3536126.
  36. 36. Bhakdi S, Tranum-Jensen J, Sziegoleit A. Mechanism of membrane damage by streptolysin-O. Infection and immunity. 1985;47(1):52–60. Epub 1985/01/01. pmid:3880730; PubMed Central PMCID: PMC261464.
  37. 37. Timmer AM, Timmer JC, Pence MA, Hsu LC, Ghochani M, Frey TG, et al. Streptolysin O promotes group A Streptococcus immune evasion by accelerated macrophage apoptosis. The Journal of biological chemistry. 2009;284(2):862–71. Epub 2008/11/13. pmid:19001420; PubMed Central PMCID: PMC2613605.
  38. 38. Bricker AL, Cywes C, Ashbaugh CD, Wessels MR. NAD+-glycohydrolase acts as an intracellular toxin to enhance the extracellular survival of group A streptococci. Molecular microbiology. 2002;44(1):257–69. Epub 2002/04/23. pmid:11967084.
  39. 39. Ashbaugh CD, Warren HB, Carey VJ, Wessels MR. Molecular analysis of the role of the group A streptococcal cysteine protease, hyaluronic acid capsule, and M protein in a murine model of human invasive soft-tissue infection. The Journal of clinical investigation. 1998;102(3):550–60. Epub 1998/08/06. pmid:9691092; PubMed Central PMCID: PMC508916.
  40. 40. Courtney HS, Hasty DL, Dale JB. Anti-phagocytic mechanisms of Streptococcus pyogenes: binding of fibrinogen to M-related protein. Molecular microbiology. 2006;59(3):936–47. Epub 2006/01/20. pmid:16420362.
  41. 41. Lancefield RC. Current knowledge of type-specific M antigens of group A streptococci. Journal of immunology (Baltimore, Md: 1950). 1962;89:307–13. Epub 1962/09/01. pmid:14461914.
  42. 42. Chiarot E, Faralla C, Chiappini N, Tuscano G, Falugi F, Gambellini G, et al. Targeted amino acid substitutions impair streptolysin O toxicity and group A Streptococcus virulence. mBio. 2013;4(1):e00387–12. Epub 2013/01/10. pmid:23300245; PubMed Central PMCID: PMC3546560.
  43. 43. Dale JB, Chiang EY, Hasty DL, Courtney HS. Antibodies against a synthetic peptide of SagA neutralize the cytolytic activity of streptolysin S from group A streptococci. Infection and immunity. 2002;70(4):2166–70. Epub 2002/03/16. pmid:11895983; PubMed Central PMCID: PMC127879.
  44. 44. Rivera-Hernandez T, Carnathan DG, Jones S, Cork AJ, Davies MR, Moyle PM, et al. An Experimental Group A Streptococcus Vaccine That Reduces Pharyngitis and Tonsillitis in a Nonhuman Primate Model. mBio. 2019;10(2). Epub 2019/05/02. pmid:31040243; PubMed Central PMCID: PMC6495378.
  45. 45. Holm SE, Norrby A, Bergholm AM, Norgren M. Aspects of pathogenesis of serious group A streptococcal infections in Sweden, 1988–1989. The Journal of infectious diseases. 1992;166(1):31–7. Epub 1992/07/01. pmid:1607705.
  46. 46. Norrby-Teglund A, Pauksens K, Holm SE, Norgren M. Relation between low capacity of human sera to inhibit streptococcal mitogens and serious manifestation of disease. The Journal of infectious diseases. 1994;170(3):585–91. Epub 1994/09/01. pmid:8077715.
  47. 47. Norrby-Teglund A, Kaul R, Low DE, McGeer A, Andersson J, Andersson U, et al. Evidence for the presence of streptococcal-superantigen-neutralizing antibodies in normal polyspecific immunoglobulin G. Infection and immunity. 1996;64(12):5395–8. Epub 1996/12/01. pmid:8945593; PubMed Central PMCID: PMC174535.
  48. 48. Mehta S, McGeer A, Low DE, Hallett D, Bowman DJ, Grossman SL, et al. Morbidity and mortality of patients with invasive group A streptococcal infections admitted to the ICU. Chest. 2006;130(6):1679–86. Epub 2006/12/15. pmid:17166982.
  49. 49. Norrby-Teglund A, Kaul R, Low DE, McGeer A, Newton DW, Andersson J, et al. Plasma from patients with severe invasive group A streptococcal infections treated with normal polyspecific IgG inhibits streptococcal superantigen-induced T cell proliferation and cytokine production. Journal of immunology (Baltimore, Md: 1950). 1996;156(8):3057–64. Epub 1996/04/15. pmid:8609429.
  50. 50. Darenberg J, Soderquist B, Normark BH, Norrby-Teglund A. Differences in potency of intravenous polyspecific immunoglobulin G against streptococcal and staphylococcal superantigens: implications for therapy of toxic shock syndrome. Clinical infectious diseases: an official publication of the Infectious Diseases Society of America. 2004;38(6):836–42. Epub 2004/03/05. pmid:14999628.
  51. 51. Cawley MJ, Briggs M, Haith LR Jr., Reilly KJ, Guilday RE, Braxton GR, et al. Intravenous immunoglobulin as adjunctive treatment for streptococcal toxic shock syndrome associated with necrotizing fasciitis: case report and review. Pharmacotherapy. 1999;19(9):1094–8. Epub 1999/12/28. pmid:10610017.
  52. 52. Basma H, Norrby-Teglund A, McGeer A, Low DE, El-Ahmedy O, Dale JB, et al. Opsonic antibodies to the surface M protein of group A streptococci in pooled normal immunoglobulins (IVIG): potential impact on the clinical efficacy of IVIG therapy for severe invasive group A streptococcal infections. Infection and immunity. 1998;66(5):2279–83. Epub 1998/05/09. pmid:9573118; PubMed Central PMCID: PMC108192.
  53. 53. Kaul R, McGeer A, Norrby-Teglund A, Kotb M, Schwartz B, O'Rourke K, et al. Intravenous immunoglobulin therapy for streptococcal toxic shock syndrome—a comparative observational study. The Canadian Streptococcal Study Group. Clinical infectious diseases: an official publication of the Infectious Diseases Society of America. 1999;28(4):800–7. Epub 2000/05/29. pmid:10825042.
  54. 54. Parks T, Wilson C, Curtis N, Norrby-Teglund A, Sriskandan S. Polyspecific Intravenous Immunoglobulin in Clindamycin-treated Patients With Streptococcal Toxic Shock Syndrome: A Systematic Review and Meta-analysis. Clinical infectious diseases: an official publication of the Infectious Diseases Society of America. 2018;67(9):1434–6. Epub 2018/05/23. pmid:29788397; PubMed Central PMCID: PMC6186853.
  55. 55. Alejandria MM, Lansang MA, Dans LF, Mantaring JB 3rd. Intravenous immunoglobulin for treating sepsis, severe sepsis and septic shock. The Cochrane database of systematic reviews. 2013;(9):Cd001090. Epub 2013/09/18. pmid:24043371; PubMed Central PMCID: PMC6516813.
  56. 56. Kadri SS, Swihart BJ, Bonne SL, Hohmann SF, Hennessy LV, Louras P, et al. Impact of Intravenous Immunoglobulin on Survival in Necrotizing Fasciitis With Vasopressor-Dependent Shock: A Propensity Score-Matched Analysis From 130 US Hospitals. Clinical infectious diseases: an official publication of the Infectious Diseases Society of America. 2017;64(7):877–85. Epub 2016/12/31. pmid:28034881; PubMed Central PMCID: PMC5850528.
  57. 57. Norrby-Teglund A, Basma H, Andersson J, McGeer A, Low DE, Kotb M. Varying titers of neutralizing antibodies to streptococcal superantigens in different preparations of normal polyspecific immunoglobulin G: implications for therapeutic efficacy. Clinical infectious diseases: an official publication of the Infectious Diseases Society of America. 1998;26(3):631–8. Epub 1998/04/03. pmid:9524835.
  58. 58. Reglinski M, Gierula M, Lynskey NN, Edwards RJ, Sriskandan S. Identification of the Streptococcus pyogenes surface antigens recognised by pooled human immunoglobulin. Scientific reports. 2015;5:15825. Epub 2015/10/29. pmid:26508447; PubMed Central PMCID: PMC4623672.
  59. 59. Svenson M, Hansen MB, Bendtzen K. Binding of cytokines to pharmaceutically prepared human immunoglobulin. The Journal of clinical investigation. 1993;92(5):2533–9. Epub 1993/11/01. pmid:8227366; PubMed Central PMCID: PMC288439.
  60. 60. Balthasar SXW-LaJP. Pharmacokinetic and Pharmacodynamic Considerations for the Use of Monoclonal Antibodies in the Treatment of Bacterial Infections. Antibodies. 2018;7(5).
  61. 61. Kawabata S, Kunitomo E, Terao Y, Nakagawa I, Kikuchi K, Totsuka K, et al. Systemic and mucosal immunizations with fibronectin-binding protein FBP54 induce protective immune responses against Streptococcus pyogenes challenge in mice. Infection and immunity. 2001;69(2):924–30. Epub 2001/02/13. pmid:11159987; PubMed Central PMCID: PMC97971.
  62. 62. Huang YS, Fisher M, Nasrawi Z, Eichenbaum Z. Defense from the Group A Streptococcus by active and passive vaccination with the streptococcal hemoprotein receptor. The Journal of infectious diseases. 2011;203(11):1595–601. Epub 2011/05/20. pmid:21592989; PubMed Central PMCID: PMC3096790.
  63. 63. Zabriskie JB, Poon-King T, Blake MS, Michon F, Yoshinaga M. Phagocytic, serological, and protective properties of streptococcal group A carbohydrate antibodies. Advances in experimental medicine and biology. 1997;418:917–9. Epub 1997/01/01. pmid:9331798.
  64. 64. Pandey M, Batzloff MR, Good MF. Mechanism of protection induced by group A Streptococcus vaccine candidate J8-DT: contribution of B and T-cells towards protection. PloS one. 2009;4(4):e5147. Epub 2009/04/03. pmid:19340309; PubMed Central PMCID: PMC2660439.
  65. 65. Lannergard J, Gustafsson MC, Waldemarsson J, Norrby-Teglund A, Stalhammar-Carlemalm M, Lindahl G. The Hypervariable region of Streptococcus pyogenes M protein escapes antibody attack by antigenic variation and weak immunogenicity. Cell host & microbe. 2011;10(2):147–57. Epub 2011/08/17. pmid:21843871.
  66. 66. Penfound TA, Chiang EY, Ahmed EA, Dale JB. Protective efficacy of group A streptococcal vaccines containing type-specific and conserved M protein epitopes. Vaccine. 2010;28(31):5017–22. Epub 2010/06/16. pmid:20546830; PubMed Central PMCID: PMC2906646.
  67. 67. Liu M, Zhu H, Zhang J, Lei B. Active and passive immunizations with the streptococcal esterase Sse protect mice against subcutaneous infection with group A streptococci. Infection and immunity. 2007;75(7):3651–7. Epub 2007/05/16. pmid:17502395; PubMed Central PMCID: PMC1932925.
  68. 68. Zeppa JJ, Kasper KJ, Mohorovic I, Mazzuca DM, Haeryfar SMM, McCormick JK. Nasopharyngeal infection by Streptococcus pyogenes requires superantigen-responsive Vbeta-specific T cells. Proceedings of the National Academy of Sciences of the United States of America. 2017. Epub 2017/08/11. pmid:28794279; PubMed Central PMCID: PMC5617250.
  69. 69. Bessen D, Fischetti VA. Passive acquired mucosal immunity to group A streptococci by secretory immunoglobulin A. The Journal of experimental medicine. 1988;167(6):1945–50. Epub 1988/06/01. pmid:3290383; PubMed Central PMCID: PMC2189674.
  70. 70. Park HS, Cleary PP. Active and passive intranasal immunizations with streptococcal surface protein C5a peptidase prevent infection of murine nasal mucosa-associated lymphoid tissue, a functional homologue of human tonsils. Infection and immunity. 2005;73(12):7878–86. Epub 2005/11/22. pmid:16299278; PubMed Central PMCID: PMC1307028.
  71. 71. Shapiro SZ. Lessons for general vaccinology research from attempts to develop an HIV vaccine. Vaccine. 2019;37(26):3400–8. Epub 2019/04/14. pmid:30979571.
  72. 72. Poland GA. Influenza vaccine failure: failure to protect or failure to understand? Expert review of vaccines. 2018;17(6):495–502. Epub 2018/06/09. pmid:29883218; PubMed Central PMCID: PMC6330882.
  73. 73. Holmes SJ, Granoff DM. The biology of Haemophilus influenzae type b vaccination failure. The Journal of infectious diseases. 1992;165 Suppl 1:S121–8. Epub 1992/06/01. pmid:1588145.
  74. 74. Oligbu G, Hsia Y, Folgori L, Collins S, Ladhani S. Pneumococcal conjugate vaccine failure in children: A systematic review of the literature. Vaccine. 2016;34(50):6126–32. Epub 2016/11/14. pmid:27838066.