Immunization of cows with novel core glycolipid vaccine induces anti-endotoxin antibodies in bovine colostrum

Vaccine 32 (2014) 6107–6114

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Immunization of cows with novel core glycolipid vaccine induces anti-endotoxin antibodies in bovine colostrum Alan S. Cross a,∗ , Hubert J. Karreman b , Lei Zhang a , Zeil Rosenberg c , Steven M. Opal d , Andrew Lees e a

Center for Vaccine Development, University of Maryland School of Medicine, 685 West Baltimore Street, HSF 1, Suite 480, Baltimore 21201, United States The Rodale Institute, 611 Siegfriedale Road, Kutztown, PA 19530, United States c Bali BioSciences, United States d Brown University, Alpert School of Medicine, Providence, RI, United States e Fina Biosolutions LLC, Rockville, MD, United States b

a r t i c l e

i n f o

Article history: Received 31 March 2014 Received in revised form 25 August 2014 Accepted 26 August 2014 Available online 19 September 2014 Keywords: Bovine colostrum Lipopolysaccharide Endotoxemia Anti-core endotoxin antibody

a b s t r a c t Background: Translocation of gut-derived Gram-negative bacterial (GNB) lipopolysaccharide (LPS, or endotoxin) is a source of systemic inflammation that exacerbates HIV, cardiovascular and gastrointestinal diseases and malnutrition. The oral administration of bovine colostrum (BC) reduces endotoxemia in patients with impaired gut barrier function. Consequently, BC enriched in antibodies to LPS may ameliorate endotoxemia-related morbidities. We developed a detoxified J5 LPS/group B meningococcal outer membrane protein (J5dLPS/OMP) vaccine that induces antibodies against a highly conserved core region of LPS and protects against heterologous GNB infection. We now examine the ability of this vaccine to elicit anti-core endotoxin antibodies in BC. Methods: Two cohorts of pregnant cows were immunized with this vaccine in combination with FICA (Cohort 1) or Emulsigen-D® (Cohort 2) adjuvants. Antibody responses to the J5 core LPS antigen were measured in both serum and colostrum and compared to antibody levels elicited by a commercially available veterinary vaccine (J5 Bacterin® ) comprised of heat-killed Escherichia coli O111, J5 mutant bacteria, from which the J5 LPS was purified. Results: The J5dLPS/OMP vaccine induced high titers of serum IgG antibody to J5 LPS in all seven cows. Both IgG and to a lesser extent IgA anti-J5 LPS antibodies were generated in the colostrum. The J5dLPS/OMP vaccine was significantly more immunogenic in mice than was the J5 Bacterin® . BC enriched in anti-J5 LPS antibody reduced circulating endotoxin levels in neutropenic rats, a model of “leaky gut”. Conclusion: The J5dLPS/OMP vaccine elicits high titers of serum anti-endotoxin antibodies in cows that is passed to the colostrum. This BC enriched in anti-core LPS antibodies has the potential to reduce endotoxemia and ameliorate endotoxin-related systemic inflammation in patients with impaired gut barrier function. Since this vaccine is significantly more immunogenic than the J5 Bacterin® vaccine, this J5dLPS/OMP vaccine might prove to be more useful for veterinary indications as well. © 2014 Elsevier Ltd. All rights reserved.

1. Introduction Human colostrum, the first milk of mothers, is enriched in nutrients and non-specific immune factors that provide passive immunity until newborn immunity is established [1]. As is the case

∗ Corresponding author at: Center for Vaccine Development, University of Maryland School of Medicine, 685 West Baltimore Street, HSF 1, Suite 480, Baltimore, MD 21201, United States. Tel.: +1 410 706 5328; fax: +1 410 706 6205. E-mail addresses: [email protected] (A.S. Cross), [email protected] (H.J. Karreman), [email protected] (L. Zhang), [email protected] (Z. Rosenberg), Steven [email protected] (S.M. Opal), [email protected] (A. Lees). http://dx.doi.org/10.1016/j.vaccine.2014.08.083 0264-410X/© 2014 Elsevier Ltd. All rights reserved.

for human colostrum, bovine colostrum (BC) is rich in immunoglobulins, antibacterial peptides, lactoferrin, cytokines and nutrients [2]. Bovine IgG1, the main milk antibody, survives transit through the gut, remains active in the intestinal tract and may replace the need for secretory IgA. The safety and efficacy of BC in treating diarrheal infection in humans, including children is well-established [3–8]. The gut is a major site of microbial colonization. Increases in gut permeability leading to systemic microbial translocation and Gram-negative bacterial (GNB) endotoxemia play a critical role in the morbidity/mortality of many conditions. Endotoxemia during coronary artery bypass graft (CABG) surgery is associated with increased morbidity [9,10]. Translocation of endotoxin

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from the gut correlates with the severity of HIV infection, and contributes to the loss of CD4+ T cells, increased viral load and innate and adaptive immune dysfunction [11–13]. Chronic HIV infection is characterized by pathologic systemic activation of the immune system, presumably due to endotoxemia [12]. During malnutrition, gut-derived endotoxemia impairs immune cell function leading to recurrent infections, accelerates the development of AIDS in HIV-infected malnourished children and impedes growth [14,15]. We developed a vaccine that induces broadly cross-reactive anti-endotoxin antibodies that recognize Pseudomonas aeruginosa and Enterobacteriaceae [16]. This vaccine is comprised of the lipopolysaccharide (LPS) of a strain of Escherichia coli (E. coli O111: H4, J5 (Rc) mutant) that lacks an O polysaccharide, thereby exposing the conserved LPS core to the immune system. Alkali treatment of the J5 LPS removes (“detoxifies”) the ester-linked fatty acids which reduces the endotoxicity and therefore the reactogenicity of the vaccine, while preserving its immunogenicity. The detoxified J5LPS (J5dLPS) is non-covalently complexed to a group B meningococcal outer membrane protein (OMP) which improves the immunogenicity of the vaccine. Animals exposed to this J5dLPS/OMP complex vaccine mount a protective antibody response in multiple models of infection and is well-tolerated in humans [17–19]. Since oral BC improves gut function and reduces endotoxemia, in this pilot study we asked whether this J5dLPS vaccine can elicit enhanced levels of colostral antibodies against a broad range of GNB endotoxins as a potential adjunctive therapy in human illnesses associated with translocation of endotoxin into the systemic circulation from a “leaky” gut and the associated endotoxin-related morbidities. While an existing whole cell J5 vaccine (J5 Bacterin® ) is used in the dairy industry to prevent bovine mastitis, it has not been used to enrich BC with anti-endotoxin antibodies for use as therapy in humans.

sides of the neck with different doses of vaccine given in conjunction with either Freund’s Incomplete Adjuvant (FICA) (Cohort 1) or Emulsigen-D® (Cohort 2), an established veterinary oil-in-water adjuvant. 2.3. Murine immunizations In order to compare the immunogenicity of the J5dLPS/OMP vaccine with the commercially-available J5 Bacterin® vaccine (Poultry Health Labs, Division of PHL Associates, Inc., Davis, CA – now Zoetis J5), we immunized outbred ICR mice (6–8 week old females, Charles River) intraperitoneally at days 0, 14 and 28 in a volume of 200 ␮l under a protocol approved by the IACUC of the University of Maryland, Baltimore. Mice received PBS, J5 Bacterin® (∼2 × 108 CFU heat-killed whole bacteria based on manufacturer’s data sheet) or J5dLPS/OMP vaccine (22 ␮g J5dLPS) and were bled retro-orbitally 7 days after the third immunization. 2.4. ELISA assay We adapted our previously described ELISA assay to measure the antibody levels in the serum, milk and colostrum of cows and serum of mice [16]. For some studies J5 Bacterin® was used as capture antigen. The amount of antibody was expressed in optical density units (ODU) which was derived from the optical density reading from the linear portion of the ELISA curve (usually at an OD ∼0.2) multiplied by the dilution at which that reading was obtained. 2.5. Reduction of endotoxemia in neutropenic rat model of “leaky gut”

Two cohorts of cows were selected based on their expectation to give birth within 3 months and immunized in this study. Both cohorts were from organically-maintained herds in rural Pennsylvania for whom one of us (HK) served as veterinarian (Table 1).

Specific pathogen-free, female, Sprague-Dawley rats (Charles River Laboratories; 150–200 g, BW) were rendered neutropenic using our standard protocol and approved by the Brown University IACUC [19]. The treatment group received the hyperimmune colostrum from either cow 1b or cow 1c by orogastric feeding while the negative control group was given either saline or cow’s milk. Following oral challenge with P. aeruginosa, rats typically develop fever and endotoxemia starting at day 5 after infection. Endotoxin levels were measured at baseline and at onset of fever from heattreated plasma samples using the quantitative Limulus Amebocyte Lysate (LAL, Associates of Cape Cod, Falmouth, MA) assay using standard methods [19].

2.2. Immunization

2.6. Statistics

2.2.1. Vaccine This vaccine was made under non-GMP conditions as previously described [16]. Briefly, E. coli J5 LPS was purchased from List Biologics (Campbell, CA) and treated with 0.2 M NaOH for 150 ± 10 min, and later neutralized with 1 M acetic acid. Group B meningococcal OMP was a gift from Dr. Kenneth Eckels at the Bioproduction Pilot Facility of the Walter Reed Army Institute of Research. The J5dLPS and OMP were mixed in a ratio of 1:1.2 (w/w LPS/OMP). Vials of vaccine were filled at either 440 or 110 ␮g of J5dLPS/ml.

Antibody levels were compared by the Mann–Whitney test (two-tailed) for non-parametric groups, unless otherwise stated.

2. Methods 2.1. Cows

2.2.2. Bovine immunization and sample collections Milk was obtained at dry-off (i.e. the end of the preceding lactation) from all four quarters of each cow’s udder as a preimmunization control. Blood was obtained from the coccygeal vein for serum antibody determinations before each of four immunizations and 8 days after the fourth immunization (Cohort 1). In Cohort 2 blood was obtained before each of three immunizations and at day 7 post-final immunization. Colostrum was obtained from each cow within a day of delivery and for 5 days thereafter. Cattle were immunized subcutaneously at alternating sites on the left and right

3. Results 3.1. Reactogenicity Each of the cows tolerated the immunizations well; however, one of the cows in Cohort 1 had an indurated area 3 cm in diameter over each of the immunization sites that persisted for the entire 6 week observation period. In contrast, in Cohort 2 Emulsigen-D® did not result in any induration beyond the typical time (∼48 h) for absorption from a subcutaneous injection. 3.2. Immunization with the J5dLPS/OMP vaccine induces a serum antibody response Following the second immunization of Cohort 1 at 4 weeks there was a 2.5–7.5-fold increase in IgG antibody levels over baseline (Fig. 1A and B). This increased slightly after the third immunization

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Table 1 Characteristics of the two cohorts of cows immunized. Breed

Age

Previous deliveries

Dose

Last dose (in days) before calving

Cohort 1a 1a 2b 3c

Jersey/Holstein Jersey/Holstein Holstein

8 8 5

6 6 3

440 ␮g 440 ␮g 1760 ␮g

8 44 16

Cohort 2b 2a 2b 2c 2d

Holstein Holstein Holstein Holstein

4 4 1 2

3 3 0 0

1100 ␮g 275 ␮g 275 ␮g 1100 ␮g

17 37 44 11

a Cows were immunized subcutaneously at alternating sites on Day 0, Day 16, Day 41 and Day 58 with the indicated dose of vaccine in 4 ml + 4 ml of FICA (total 8 ml/dose). (On Day 58, the cows were given double the dose without FICA in the same 8 ml volume). Each cow was vaccinated 1.5 yrs earlier with Scourgard 4KC® which has a coliform component, but at the time of J5dLPS/OMP immunization was at the end of any expected protection. b Cows were immunized subcutaneously at alternating sites on Day 0, Day 18 and Day 43 with the indicated dose of vaccine in 2.4 ml + 0.6 ml of Emulsigen-D® (total 3.0 ml/dose). All were previously immunized with Bovishield® which contains bovine viral diarrhea virus types 1 and 2, bovine respiratory syncytial virus, parainfluenza 3 virus, infectious bovine rhinotracheitis virus and leptospira antigens, but none received any coliform-containing vaccine.

in two of the cows and markedly in the third. A fourth immunization in the absence of adjuvant at 9 weeks resulted in a further increase in antibody level. (Note difference in ODU vs. fold-increase for cows 1b and 1c – probably representing differences in baseline antibody levels). Each of the four cows in Cohort 2 mounted a robust serum antibody response after the third immunization (Fig. 1C and D), although the response of cow 2d was not as great as the other three. In three of the four cows there was a marked increase in IgG over baseline after the second immunization (except for cow 2d), which increased further after the 3d immunization. Both serum

IgG1and IgG2 levels increased with immunization in each cohort (Supplementary Fig. 1). 3.3. Colostral anti-J5 LPS antibody response In Cohort 1 we compared the level of anti-J5 IgG in the milk at dry-off to the anti-J5 IgG level in colostrum. There was a 12 to nearly 370-fold higher level of colostral anti-J5 LPS IgG compared to levels in milk, while there was a 9–22-fold higher level of colostral IgA and 9–95-fold higher level of colostral IgM (Fig. 2). J5dLPS

Fig. 1. Serum IgG immune response of cows to J5dLPS/OMP vaccine. Cows in Cohort 1 (panels A & B) and Cohort 2 (panels C and D) were immunized with vaccine and either FICA (Cohort 1) or Emulsigen-D (Cohort 2) according to the schedule described in Table 1. Serum was collected before each immunization and 7–8 days after the final immunization. Data are expressed in Optical Density Units (ODU) (see Section 2) (panels A and C) and fold-increase of post-immune sera compared to pre-immune anti-J5 LPS levels (panels B & D).

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with similar levels of colostral IgG (cows 2b and 2d) had nearly a 6-fold difference in serum IgG. Each of the cows expressed J5LPS-specific IgA in the colostrum (Fig. 3B). Since there is a licensed anti-endotoxin vaccine made from the same parent bacterial strain as our J5dLPS/OMP vaccine, we examined whether the J5dLPS/OMP vaccine elicited serum antibodies to J5 Bacterin® . Immunization of cows with the J5dLPS/OMP vaccine induced robust levels of serum antibody against J5 Bacterin® antigen (Supplementary Fig. 2). 3.4. Comparison of immunogenicity of Bacterin® to that of J5dLPS/OMP in mice J5 Bacterin® is frequently administered to cows for protection against bovine mastitis. Assuming that the J5 LPS is the active immunogen in the J5 Bacterin® , the J5dLPS/OMP vaccine has about 40 times the LPS content per dose as the J5 Bacterin® (22 ␮g vs. ∼0.5 ␮g, respectively) [20]. Immunization of mice with J5 Bacterin® at the 2 week intervals induced anti-J5 LPS IgG antibody levels between 649 and 11,600 ODU (mean 2883 ± 3872) while administration of the J5 dLPS/OMP vaccine at the same intervals resulted in a >5-fold greater mean IgG antibody level (Fig. 4, panel A). J5dLPS/OMP immunization elicited antibody levels against J5 Bacterin® that were lower than those observed with J5 Bacterin® immunization, but the differences were not significant (Fig. 4, panel B). 3.5. Functional antibody response Administration of hyperimmune BC reduced endotoxemia at the onset of fever in an animal model of “leaky gut”, a condition often encountered clinically. Neutropenic rats challenged with P. aeruginosa developed fever by day 5, indicative of systemic infection [19]. Rats were given either J5dLPS/OMP antibody-enriched BC on days 3 and 5, or either milk or saline as controls. Rats that received BC enriched in anti-J5dLPS/OMP had lower circulating endotoxin levels at the onset of fever than those that received control treatment (Fig. 5). 4. Discussion

Fig. 2. Comparison of immunoglobulins G, A and M in pre-immunization milk (obtained at dry-off) to colostrum (obtained within first day of delivery). Milk was obtained at dry-off and colostrum within first day of delivery for Cohort #1 and isotype-specific antibody to J5 LPS determined by ELISA. The anti-J5 LPS IgG, IgA and IgM antibody levels were determined by ELISA and expressed as ODU.

immunization elicited a 8-fold increase in anti-J5 LPS IgG colostral antibody level compared to the J5 LPS IgG antibody level in the colostrum of a non-immunized cow (data not shown). In comparing the levels of anti-J5 immunoglobulin in the colostrum to that of serum, the cow (1b) with the greatest levels of colostral IgA and IgM (latter not shown) antibody did not have the greatest colostral IgG response (Fig. 3A). The colostral anti-J5 LPS IgG was increased above the serum level in two of the cows (cows 1a and 1c) and lower than the serum IgG level in another (cow 1b) (Fig. 3A). Similar to Cohort 1, the J5 LPS IgG responses in the colostrum of cows in Cohort 2 did not appear to follow any pattern with regard to the IgG level in the serum. The cow with the greatest colostral IgG level (cow 2a) had a level of serum IgG antibody similar to that of 2c, whose colostral IgG levels were nearly 10-fold lower (Fig. 3B). Cows

Bovine colostrum enriched in antibodies against enteric pathogens protects against diarrheal infections in both children and adults [3–8]. Consequently, we speculated that BC enriched in antibodies against GNB LPS may be a useful adjunct in the treatment of clinical conditions characterized by endotoxemia. This study shows that immunization of cows (both heifers and multi-parous) with the J5dLPS/OMP vaccine with adjuvant during the last trimester of pregnancy results in robust serum and colostral IgG antibody responses to the J5 LPS. The serum IgG levels were not necessarily predictive of the likelihood of detecting high levels of colostral IgG. These data indicate that it is possible to induce high-titered anti-core endotoxin antibody levels in BC for oral administration to humans. Finally, in a pilot study BC enriched in anti-endotoxin antibodies reduced the level of endotoxemia in rats with systemic inflammatory manifestations. The J5 (Rc) mutant of E. coli O111:B4 lacks a gal-epimerase by which the organism links the O-polysaccharide to its core, thereby exposing the relatively conserved core to the immune system. A whole, heat-killed J5 bacterial vaccine elicited antibodies that recognized heterologous GNB and provided passive protection in animal models of sepsis as well as in clinical trials in human subjects when given either passively as therapy or prophylactically in an intensive care unit setting [21,22]. The killed J5 bacterial immunogen was not further developed into a vaccine for human use, however.

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A

6111

B

IgG 125000

75000

Serum Colostrum

ODU

ODU

50000

25000

Serum Colostrum

100000 75000 50000 25000

0 1a

1b

0

1c

2a

Cow

Colostrum Cow

IgA

2c

2d

Cow Serum

IgG

2b

IgG Ratio

IgG

1a

1,090

10,444

3,597

2.90

1b

10,108

15,686

28,748

0.55

1c

1,581

70,320

10,444

6.73

Cow 2a 2b 2c 2d

Colostrum Serum IgG Ratio IgA IgG IgG 37,102 4.37 3,880 162,300 2,595 13,320 60,840 0.22 2,100 18,940 41,084 0.46 2,250 10,800 9,725 1.11

Fig. 3. Anti-J5 LPS antibodies in the serum and colostrum following immunization with J5dLPS/OMP vaccine. The level of IgG antibody in the serum obtained 2 weeks after the third immunization was compared to the anti-J5 LPS antibody levels in the colostrum obtained within one day of delivery in cows in Cohort 1 (panel A) and Cohort 2 (panel B). The levels of colostral IgA and IgG as well as serum IgG levels in Cohorts 1 (panel A) and 2 (panel B) are expressed as ODU. The colostral IgG/serum IgG ratio was determined.

With active immunization or passive administration of vaccineinduced antibodies, this J5dLPS/OMP vaccine is highly protective against lethal infection caused by heterologous bacteria in different animal models of sepsis [17–19] and has been well-tolerated in a phase 1 clinical trials in human subjects [23]. We now report that this vaccine induces robust serum and colostral anti-endotoxin antibody responses in cows, with the latter levels in some instances exceeding those found in serum (Fig. 3). Thus, this vaccine may be used to generate colostrum enriched in anti-core endotoxin antibodies. Many clinical conditions, such a hemorrhagic trauma, HIV infection, chemotherapy, radiation, inflammatory bowel disease, burns, strenuous exercise and heat stroke, are associated with increased gut permeability with translocation of bacteria into the blood

stream resulting in endotoxemia [11,24–28]. For example, endotoxemia has been proposed as an important cause of postoperative morbidity and mortality after elective CABG [9,10] as well as a contributor to congestive heart failure [29]. Further, low levels of antibody to the core structure of LPS (EndoCAb® ) before CABG surgery is an independent predictor of an adverse postoperative outcome, is associated with increased mortality up to 5 yrs later [30]. In other studies, nearly 60% of patients had evidence of endotoxemia upon admission to the ICU and the degree of endotoxin activity correlated with the likelihood of severity of sepsis, organ failure and outcome [31]. BC preparations enriched in antiendotoxin antibodies similar to those measured by the EndoCab® assay may offer additional benefit by neutralizing post-operative endotoxemia, and perhaps helping to restore gut barrier function.

Fig. 4. Immunization of mice with J5 Bacterin® vs. J5dLPS/OMP vaccine. After obtaining pre-immunization sera from retro-orbital bleeds, outbred CD-1 mice (n = 7/group) were immunized intraperitoneally three times at 2 week intervals with 200 ␮l of either J5 Bacterin® (∼2 × 108 CFU) or J5dLPS/OMP vaccine (22 ␮g J5dLPS)). Sera obtained 14 days after the third bleed were assayed for antibody levels to J5 dLPS (panel A) and to J5 Bacterin® (panel B). The serum levels of anti-J5 LPS IgG were greater in mice immunized with the J5dLPS/OMP vaccine than with the J5 Bacterin vaccine (p < 0.02 by ANOVA and Student t test). There was no significant difference in IgG antibody levels elicited by the two vaccines to J5 Bacterin® .

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n g / ml

10

1

0. 1 1b

1c

Control p= . 027

Fig. 5. Endotoxemia is reduced in neutropenic rats pretreated with J5 LPS hyperimmune colostrum compared to an equal volume of milk. Rats were rendered neutropenic with cyclophosphamide and treated with moxafloxacin to overcome colonization resistance before oral infection with P. aeruginosa (106 CFU on days 0, 2 and 4) as previously described [19]. Rats were administered colostrum by orogastric feeding (2/ml/day on days 3 and 5) from either cow 1b (n = 8) or 1c (n = 8) or an equal volume of cow’s milk or saline as negative control (n = 16). The circulating endotoxin levels were measured from plasma samples sterilely obtained at baseline (day-2) and day 5 following infection and measured by a quantitative turbidometric LAL assay as we previously described [19]. The circulating endotoxin levels at day 5 after infection (onset of fever) are represented as the median values with 95% confidence intervals and analyzed by the Kruskal–Wallis method for non-parametric data in multiple groups. Circulating endotoxin levels decreased following administration of bovine colostrum (p = 0.027).

Since the endotoxemia contributes to a persistent and deleterious inflammation, it may be desirable to examine interventions that can reduce endotoxemia. Colostrum has an important role during bacterial colonization of the gut by promoting colonization of commensal bacteria and by trapping, pathogens and viruses to prevent invasion [1,2]. BC has both nutritional components and immunologic mediators that promote gut integrity and mucosal recovery after severe diarrhea and injury (e.g. IGF-1 and TGF-␤2), immunoglobulins, immune modulators (e.g. soluble TLR2, soluble CD14) and has antimicrobial activity [2]. In addition to antimicrobial peptides, BC has proteins such as lactoferrin that has in vitro antimicrobial activity against Gram-positive and-negative bacteria, fungi and viruses (rotavirus, enterovirus and adenovirus) [32]. Bovine lactoferrin supplementation decreased the incidence of late onset sepsis in very low birth weight neonates (<1500 g), and had an even greater impact on infection and mortality in newborns < 1000 g [33,34]. The oral administration of BC reduces LPS translocation and systemic inflammation and has a record of clinical use and safety. The prophylactic enteral administration of BC for 2 days reduced the generation of post-operative acute phase reactants in patients undergoing elective CABG [35]. Similarly, the prophylactic administration of a BC preparation reduced perioperative endotoxemia after abdominal surgery, perhaps by stabilizing the gut barrier [36]. Experimentally, BC prevents intestinal damage and permeability, and bacterial translocation to other organs in a rat model of ischemia/reperfusion-injury [37]. A case-controlled study that examined the causes of moderate to severe diarrhea in over 9000 children in seven developing countries found that four pathogens, rotavirus, cryptosporidiosis, enterotoxigenic E. coli (ST-ETEC) and shigella, were associated with increased mortality and stunted growth [38]. BC has been shown to ameliorate diarrheal disease caused by these pathogens [3–8]. A tablet form of hyperimmune BC provided 90% protection compared to placebo in volunteers infected with enterotoxigenic EC(ETEC) [4] and a commercial product is now available.

Malnutrition is associated with impaired gut barrier function, endotoxemia and systemic inflammation [14,15]. Malnutritionassociated endotoxemia impairs dendritic cell function, which results in a compromised adaptive immune response, and a susceptibility to infections that accelerates the malnutrition and stunts growth [14,15]. Adequate nutrition is necessary but not sufficient to insure growth. Antibiotic administration during severe malnutrition reduces mortality and increases recovery, perhaps by decreasing the translocation of GNB into the bloodstream [39]. Through both its nutritional and immunological support, BC enriched in anti-endotoxin antibodies may break that cycle. Surprisingly, oral BC decreases the severity of viral URIs in humans, and influenza-immune BC given intranasally protects mice from experimental influenza, suggesting a potential benefit in respiratory infections as well [40,41]. The severity and mortality of HIV infection is increased by malnutrition and sCD14, a biomarker for endotoxemia, is an independent predictor of mortality in HIV infection [15,42]. Breastfeeding of neonates born to HIV-positive mothers in the first 4–6 months decreases the risk of HIV and associated infections and provides survival benefit [43]. BC is a low cost, large scale source of antibodies with broad neutralizing activity for HIV that alleviates HIV-associated diarrhea [44]. BC dramatically reduced stool frequency, increased body weight and CD4 counts in HIV-infected patients [45]. Since BC alone is effective in reducing the morbidity from HIV infection and malnutrition, a product that is enriched in anti-endotoxin antibodies may not only improve patient nutritional and immunologic status, but also reduce HIV-associated endotoxemia. In addition to the potential use of this vaccine for generating BC enriched in anti-endotoxin antibodies, this vaccine may be useful in preventing bovine mastitis, a devastating infection in cows. A killed bacterial formulation of this vaccine has been widely used in the dairy industry to protect calves at risk of neonatal sepsis and for the prevention of bovine mastitis [46,47]. While this vaccine does not appear to prevent infection, cows that have been immunized with the J5 Bacterin® or those with natural levels of anti-J5 antibody have less severe episodes of the disease, with higher serum levels of anti-J5 LPS IgG correlating with a lower incidence of coliform mastitis; however, direct bacterial challenge experiments in J5 Bacterin® immunized cows have failed to show a consistent benefit [46,47]. J5 Bacterin® is a weak immunogen requiring at least five immunizations to achieve an elevation in IgG1 and IgG2 levels indicative of a mature immunoglobulin response [48]. If the protection is related to the level of anti-J5 LPS antibodies achieved, then the use of a more potent vaccine may show a more consistent benefit. The J5dLPS/OMP vaccine generated high-titered antibody to the J5 Bacterin® antigen in the two cohorts of cows. (Supplementary Fig. 2), and when given at equal volumes was more immunogenic in mice than J5 Bacterin® (Fig. 4). This may be due to the fact that the J5dLPS/OMP vaccine presents the relevant J5 LPS antigen at a higher concentration and perhaps in a more accessible format than the whole bacterial vaccine. A vaccine comprised of detoxified J5 LPS conjugated to chicken serum albumen delivered 2.8 mg J5dLPS/dose in conjunction with FICA. The IgG and IgM antibody titers to J5 whole cell and J5 LPS antigens were comparable with or higher than those of cows immunized with the J5 Bacterin® [49]. Recently, J5dLPS vaccines conjugated to chicken serum albumen and to hemocyanine induced anti-J5 LPS antibodies that were ≤1:3200 following immunization with 250 ␮g for each of three doses [50]. 5. Conclusion Endotoxemia occurs in multiple clinical conditions characterized by impaired gut barrier function and may result in systemic

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inflammation that complicates cardiothoracic and abdominal surgery, burns and HIV infection as well as gastrointestinal infection. In developing countries malnutrition-associated endotoxemia impairs innate and adaptive immune responses leading to recurrent infections, accelerated development of AIDS, stunted growth and further malnutrition. A different formulation of a J5 vaccine is highly immunogenic in cows, resulting in high levels of antiJ5 LPS antibodies in both serum and colostrum, which may reduce endotoxemia. Thus, a BC enriched in anti-endotoxin antibodies may be formulated into an oral safe, low-cost adjunctive therapeutic approach to reduce gut permeability, prevent endotoxemia and systemic inflammation, improve immune function, reduce diarrheal infections and reduce HIV-associated disease during periods of impaired gut barrier integrity. Acknowledgements This study was funded by the Maryland Proof-of-Concepts Alliance, University of Maryland, College Park Agreement Z855809, CFDA 12.431. We are deeply indebted to John Meulenberg (Cohort 1) and David Fisher (Cohort 2) for allowing us to study the vaccine in their cows. Barbara Boyd provided excellent administrative support for this project. Conflict of interest: Dr. Cross holds an issued patent on this vaccine and Drs. Rosenberg and Cross have filed an invention disclosure for the hyperimmune bovine colostrum. None of the other authors have a conflict of interest. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.vaccine. 2014.08.083. References [1] Walker A. Breast milk as the gold standard for protective nutrients. J Pediatr 2010;156(Suppl. 1):S3–7. [2] Stelwagen K, Carpenter E, Haigh B, Hodgkinson A, Wheeler TT. Immune components of bovine colostrum and milk. J Anim Sci 2009;87(Suppl. 1):3–9. [3] Davison GP, Daniels E, Nunan H, Moore AG, Whyte PBD, Franklin K, et al. Passive immunization of children with bovine colostrum containing antibodies to human rotavirus. Lancet 1989;2:709–12. [4] Otto W, Najnigier B, Stelmasiak T, Robins-Browne RM. Randomized control trials using a tablet formulation of hyperimmune bovine colostrum to prevent diarrhea caused by enterotoxigenic Escherichia coli in volunteers. Scand J Gastro 2011;46:862–8. [5] Huppertz H-I, Rutkowski S, Busch DH, Eisebit R, Lissner R, Karch H. Bovine colostrum ameliorates diarrhea in infection with diarrheagenic Eschericia coli, shiga-toxin-producing E. coli and E. coli expressing intimin and hemolysin. J Pediatr Gastroenterol Nutr 1999;29:452–6. [6] Tacket CO, Binion SB, Bostwick E, Losonsky G, Roy MJ, Edelman R. Efficacy of bovine milk immunoglobulin concentrate in preventing illness after Shigella flexneri challenge. Am J Trop Med Hyg 1992;47:276–83. [7] Greenberg PD, Cello JP. Treatment of severe diarrhea caused by Cryptosporidium parvum with oral bovine immunoglobulin concentrate in patients with AIDS. J Acquir Immune Defic Syndr Hum Retrovirol 1996;13:348–54. [8] Steele J, Sponseller J, Schmidt D, Cohen O, Tzipori S. Hyperimmune bovine colostrum for treatment of GI infections: a review and update on Clostridium difficile. Hum Vaccin Immunother 2013;9:1565–8. [9] Klein DJ, Briet F, Nisenbaum R, Romaschin AD, Mazer CD. Endotoxemia related to cardiopulmonary bypass is associated with increased risk of infection after cardiac surgery: a prospective observational study. Crit Care 2011;15:R69. [10] Olsen MA, Krauss M, Agniel D, Schootman M, Gentry CN, Yan Y, et al. Mortality associated with bloodstream infection after coronary artery bypass surgery. Clin Infect Dis 2008;46:1537–46. [11] Bukh AR, Meclhjorsen J, Offersen R, Jensen JMB, Toft L, Stovring H, et al. Endotoxemia is associated with altered innate and adaptive immune responses in untreated HIV-1 infected individuals. PLoS ONE 2011;6:e21275, http://dx.doi.org/10.1371/journal.pone.0021275. [12] Brenchley JM, Price DA, Schacker TW, Asher TE, Silvestri G, Rao S, et al. Microbial translocation is a cause of systemic immune activation in chronic HIV infection. Nat Med 2006;12:1365–71.

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