Evaluation of the Efficacy of a Cholera Toxin-Based <em

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1-2020

Evaluation of the E=cacy of a Cholera Toxin-Based Evaluation of the E=cacy of a Cholera Toxin-Based

Staphylococcus aureus

Vaccine Against Bovine Intramammary Vaccine Against Bovine Intramammary

Challenge Challenge

Hussain A. Alabdullah

University of Idaho

Elise Overgaard

Boise State University

Danielle Scarbrough

Boise State University

Janet E. Williams

University of Idaho

Omid Mohammad Mousa

Boise State University

See next page for additional authors

Publication Information Publication Information

Alabdullah, Hussain A.; Overgaard, Elise; Scarborough, Danielle; Williams, Janet E.; Mousa, Omid

Mohammad; Dunn, Gary; . . . and Tinker, Juliette K. (2021). Evaluation of the E=cacy of a Cholera Toxin-

Based

Staphylococcus aureus

Vaccine Against Bovine Intramammary Challenge.

Vaccines, 9

(1), 6.

https://doi.org/10.3390/vaccines9010006

Authors Authors

Hussain A. Alabdullah, Elise Overgaard, Danielle Scarbrough, Janet E. Williams, Omid Mohammad Mousa,

Gary Dunn, Laura Bond, Mark A. McGuire, and Juliette K. Tinker

This article is available at ScholarWorks: https://scholarworks.boisestate.edu/bio_facpubs/697

Article

Evaluation of the Efﬁcacy of a Cholera Toxin-Based

Staphylococcus aureus Vaccine against Bovine

Intramammary Challenge

Hussain A. Alabdullah

1,†

, Elise Overgaard

2,†

, Danielle Scarbrough

, Janet E. Williams

Omid Mohammad Mousa

, Gary Dunn

, Laura Bond

, Mark A. McGuire

and Juliette K. Tinker

2,3,



 

Citation: Alabdullah, H.A.; Overgaard,

E.; Scarbrough, D.; Williams, J.E.;

Mohammad Mousa, O.; Dunn, G.;

Bond, L.; McGuire, M.A.; Tinker, J.K.

Evaluation of the Efﬁcacy of a Cholera

Toxin-Based Staphylococcus aureus

Vaccine against Bovine Intramammary

Challenge. Vaccines 2021, 9, 6. https://

dx.doi.org/10.3390/vaccines9010006

Received: 24 November 2020

Accepted: 18 December 2020

Published: 24 December 2020

Publisher’s Note: MDPI stays neu-

tral with regard to jurisdictional claims

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censee MDPI, Basel, Switzerland. This

article is an open access article distributed

under the terms and conditions of the

Creative Commons Attribution (CC BY)

license (https://creativecommons.org/

licenses/by/4.0/).

Department of Animal and Veterinary Science, University of Idaho, Moscow, ID 83844, USA;

[email protected] (H.A.A.); [email protected] (J.E.W.); [email protected] (M.A.M.)

Biomolecular Sciences Graduate Program, Boise State University, Boise, ID 83725, USA;

eliseovergaar[email protected] (E.O.); [email protected] (D.S.)

Department of Biological Sciences, Boise State University, Boise, ID 83725, USA;

[email protected] (O.M.M.); gary[email protected] (G.D.)

Biomolecular Research Center, Boise State University, Boise, ID 83725, USA; [email protected]

* Correspondence: [email protected]

† The authors contribute equally.

Abstract:

Staphylococcus aureus (S. aureus) is a primary agent of bovine mastitis and a source of signiﬁ-

cant economic loss for the dairy industry. We previously reported antigen-speciﬁc immune induction

in the milk and serum of dairy cows following vaccination with a cholera toxin A

and B subunit

(CTA

/B) based vaccine containing the iron-regulated surface determinant A (IsdA) and clumping

factor A (ClfA) antigens of S. aureus (IsdA + ClfA-CTA

/B). The goal of the current study was to

assess the efﬁcacy of this vaccine to protect against S. aureus infection after intramammary chal-

lenge. Six mid-lactation heifers were randomized to vaccinated and control groups. On days 1 and

14 animals were inoculated intranasally with vaccine or vehicle control, and on day 20 animals were

challenged with S. aureus. Clinical outcome, milk quality, bacterial shedding, and somatic cell count

(SCC) were followed for ten days post-challenge. Vaccinated animals did not show signs of clinical

S. aureus mastitis and had lower SCCs compared to control animals during the challenge period.

Reductions in bacterial shedding were observed but were not signiﬁcant between groups. Antibody

analysis of milk and serum indicated that, upon challenge, vaccinated animals produced enhanced

IsdA- and ClfA-CTA

/B speciﬁc immunoglobulin G (IgG) responses, while responses to CTA

alone were not different between groups. Responses after challenge were largely IgG1 against the

IsdA antigen and mixed IgG1/IgG2 against the ClfA antigen. In addition, there was a signiﬁcant

increase in interferon gamma (IFN-

) expression from blood cells in vaccinated animals on day 20.

While preliminary, these ﬁndings support evidence of the induction of active immunity by IsdA +

ClfA-CTA

/B, and further assessment of this vaccine is warranted.

Keywords: Staphylococcus aureus; vaccine; bovine; mastitis

1. Introduction

Mastitis, or inﬂammation of the udder, is one of the most economically-signiﬁcant

diseases affecting dairy cattle worldwide and is most often the result of a bacterial infection.

Staphylococcus aureus (S. aureus), a main etiological agent, is highly contagious and can

spread rapidly among herds. It is estimated that up to 70% of U.S. herds are positive

for S. aureus, and this bacterium caused the highest overall annual yield losses among

other mastitis pathogens in a recent Finnish study [

]. S. aureus infections are most

commonly transmitted during the milking process and can impact animal welfare as well

as milk yield and quality [

]. The ability of this bacterium to form bioﬁlms and replicate

intracellularly can promote subclinical colonization of the mammary gland, often leading

Vaccines 2021, 9, 6. https://dx.doi.org/10.3390/vaccines9010006 https://www.mdpi.com/journal/vaccines

Vaccines 2021, 9, 6 2 of 17

to chronic infection, which is difﬁcult to detect and is frequently the source of herd re-

infection [

–

]. S. aureus is also commonly resistant to antimicrobial treatment and has

a low expected cure rate during lactation [

]. While the impact of S. aureus infection is

difﬁcult to quantify, clinical mastitis caused by Gram-positive pathogens is reported to cost

between $133 and $444 per case, or as much as USD 2 billion annually [

]. These costs

include many factors such as milk loss, veterinary expenses, diagnostic testing, and loss

of animals. Prevention of S. aureus mastitis with a cost-effective vaccine would improve

animal welfare, reduce antibiotic use, and positively impact the economics and efﬁciency

of milk production.

Previous approaches to S. aureus vaccination in cattle include whole-cell live and killed

vaccines as well as puriﬁed antigens. Currently, two whole-cell inactivated vaccines are

licensed for protection against S. aureus mastitis—Lysigin

(Boehringer Ingelheim, Duluth,

GA, USA) and Startvac

(Hipra, Girona, Spain). While efﬁcacy studies are somewhat con-

ﬂicting, these vaccines have reported moderate decreases in the incidence of new

S. aureus

intramammary infection but are not in widespread use [

–

]. Recent studies have fo-

cused on the use of multiple puriﬁed surface adhesins and secreted virulence factors to

develop a vaccine that offers more strain-to-strain cross-protection. Iron-regulated surface

determinant A (IsdA) is a ﬁbrinogen- and ﬁbronectin-binding adhesin that contributes to

iron sequestration and is a well-studied S. aureus vaccine candidate [

–

]. The presence

of isdA is conserved among bovine S. aureus, and IsdA is expressed from these strains in

milk [

–

]. The clumping factor A (ClfA) ﬁbrinogen adhesin is also highly conserved,

expressed from bovine clinical isolates, and a recognized vaccine candidate against masti-

tis [

–

]. The conservation, surface exposure, and importance in multiple mechanisms of

pathogenesis supports the inclusion of the IsdA and ClfA antigens in a multivalent bovine

vaccine. However, a number of additional antigens have been characterized and may be

necessary to protect against multiple S. aureus serotypes.

While immune correlates of protection are not known, an understanding of immune

responses is needed to inform antigen selection. The induction of both humoral and cellular

immunity is essential to combating intracellular S. aureus infection [

–

]. Cellular sub-

populations that play a central role in defense against S. aureus include neutrophils, CD8

T lymphocytes, and CD4

Th17 lymphocytes [

]. Cholera toxin (CT), produced by the

bacterium Vibrio cholerae, and the homologous heat-labile toxin I (LTI), produced by the

bacterium Escherichia coli, are gold standard vaccine adjuvants that can stimulate systemic

immunity from mucosal and dermal sites (reviewed in [

]). The mechanism of adju-

vanticity of these toxins depends upon active binding subunit targeting of dendritic cells

and neutrophils, and has been attributed to enhanced antigen presentation, upregulation

of surface molecules, and promotion of B-cell isotype switching to antigen-speciﬁc im-

munoglobulin A (IgA) and immunoglobulin G (IgG) [

–

]. CT and its non-toxic binding

subunit (CTB) can also induce Th1, Th2, and Th17 responses [42–44].

The toxic A subunit of CT (CTA) is subdivided into an enzymatically-active domain

(CTA

) and a linker domain (CTA

), which is non-covalently associated with the B subunit.

CTA

/B chimeras were ﬁrst described as a mechanism to make stable human vaccines

with antigens coupled to the CTB subunit via the A

linker domain [

]. These non-toxic

molecules retain the adjuvanticity of CTB and possess additional advantages including

ease of puriﬁcation, direct association of antigen to adjuvant, and a holotoxin-like structure

that retains binding and internalization motifs [

]. As reported previously, we have

incorporated S. aureus IsdA and ClfA into a CTA

/B vaccine platform (IsdA + ClfA-

CTA

/B). After two intranasal doses this vaccine was found to stimulate signiﬁcant S.

aureus antigen-speciﬁc humoral and cellular immunity in bovine blood and milk [49].

For this study we hypothesized that intranasal IsdA + ClfA-CTA

/B would be effective

in reducing or eliminating S. aureus shedding and disease after intramammary challenge in

cattle. We describe a preliminary trial to determine the efﬁcacy of this mucosal enterotoxin-

based vaccine to protect against acute S. aureus mastitis. While the vaccine did not prevent

bacterial shedding after challenge, results indicate that IsdA + ClfA-CTA

/B induces

Vaccines 2021, 9, 6 3 of 17

antigen-speciﬁc immune responses that may contribute to a reduction in clinical severity

and inﬁltration of leukocytes, or SCC, in infected animals.

2. Materials and Methods

2.1. Bacterial Strains, Plasmids, and Growth Conditions

S. aureus Newbould 305 was used for the cloning of isdA and clfA to construct IsdA +

ClfA-CTA

/B and was also used for bacterial challenge [

]. E. coli ClearColi

(Lucigen,

Madison, WI, USA) was used for protein expression (Table 1). The vector pARLDR19

expressing CTA

/B and containing a multiple cloning site was used to construct the

plasmids pLR001 for Isd-CTA

/B expression and pLR003 for ClfA-CTA

/B expression

(Figure 1A) as described previously [

]. For bacterial challenge, S. aureus Newbould 305

was prepared as described [

]. Brieﬂy, Newbould 305 was grown at 37

◦

C with shaking

to mid-log phase in brain–heart infusion and harvested by centrifugation at 3000

g for

15 min at 4

◦

C. The cell pellet was washed with phosphate-buffered saline (1X PBS, pH 7.2)

and adjusted to an optical density (O.D.) of 0.2 at 600 nm. Serial dilutions were performed

in 1X PBS to reach a bacterial concentration of 400 CFU/mL, as determined by plating on

blood agar (BA).

Table 1. Bacterial strains, plasmids, and primers used in this study.

Bacterial Strains Genotype or Characteristics Source

E. coli ClearColi

BL21(DE3) Lucigen, Madison, WI

S. aureus Newbould 305 Bovine clinical isolate [50]

Plasmids Gene Vector Source

pLR001 isdA (Newbould) pARLDR19 [49]

pLR003 clfA (Newbould) pARLDR19 [49]

Bovine Cytokine qPCR Primers Gene Amplicon Source

FW 5

-GCATCGTGGAGGGACTTATGA-3

GAPDH 67

[52]

RV 5

-GGGCCATCCACAGTCTTCTG-3

FW 5

-CTTGTCGGAAATGATCCAGTTTT-3

IL-10 66

[53]

RV 5

-TCAGGCCCGTGGTTCTCA-3

FW 5

-CAGAAAGCGGAAGAGAAGTCAGA-3

IFN-γ

[52]

RV 5

-TGCAGGCAGGAGGACCAT-3

FW 5

-GGCTCCCATGATTGTGGTAGTT-3

IL-6 64

[53]

RV 5

-GCCCAGTGGACAGGTTTCTG-3

Vaccines 2021, 9, x 3 of 17

enterotoxin-based vaccine to protect against acute S. aureus mastitis. While the vaccine did

not prevent bacterial shedding after challenge, results indicate that IsdA + ClfA-CTA

2/B

induces antigen-specific immune responses that may contribute to a reduction in clinical

severity and infiltration of leukocytes, or SCC, in infected animals.

2. Materials and Methods

2.1. Bacterial Strains, Plasmids, and Growth Conditions

S. aureus Newbould 305 was used for the cloning of isdA and clfA to construct IsdA +

ClfA-CTA

2/B and was also used for bacterial challenge [22,50]. E. coli ClearColi

(Lucigen,

Madison, WI, USA) was used for protein expression (Table 1). The vector pARLDR19 ex-

pressing CTA

2/B and containing a multiple cloning site was used to construct the plasmids

pLR001 for Isd-CTA

2/B expression and pLR003 for ClfA-CTA2/B expression (Figure 1A)

as described previously [51]. For bacterial challenge, S. aureus Newbould 305 was pre-

pared as described [10]. Briefly, Newbould 305 was grown at 37 °C with shaking to mid-

log phase in brain–heart infusion and harvested by centrifugation at 3000× g for 15 min at

4 °C. The cell pellet was washed with phosphate-buffered saline (1X PBS, pH 7.2) and

adjusted to an optical density (O.D.) of 0.2 at 600 nm. Serial dilutions were performed in

1X PBS to reach a bacterial concentration of 400 CFU/mL, as determined by plating on

blood agar (BA).

Table 1. Bacterial strains, plasmids, and primers used in this study.

Bacterial Strains Genotype or Characteristics Source

E. coli ClearColi

BL21(DE3) Lucigen, Madison, WI

S. aureus Newbould 305 Bovine clinical isolate [50]

Plasmids Gene Vector Source

pLR001 isdA (Newbould) pARLDR19 [49]

pLR003 clfA (Newbould) pARLDR19 [49]

Bovine Cytokine qPCR Primers Gene Amplicon Source

FW 5′-GCATCGTGGAGGGACTTATGA-3′

GAPDH 67 [52]

RV 5′-GGGCCATCCACAGTCTTCTG-3′

FW 5′-CTTGTCGGAAATGATCCAGTTTT-3′

IL-10 66 [53]

RV 5′-TCAGGCCCGTGGTTCTCA-3′

FW 5′-CAGAAAGCGGAAGAGAAGTCAGA-3′

IFN-γ 72 [52]

RV 5′-TGCAGGCAGGAGGACCAT-3′

FW 5′-GGCTCCCATGATTGTGGTAGTT-3′

IL-6 64 [53]

RV 5′-GCCCAGTGGACAGGTTTCTG-3′

Figure 1. S. aureuscholera toxin A2/B (CTA2/B) chimeric mucosal vaccines. (A) pLR001 for expres-

sion of IsdA-CTA2/B, and pLR003 for expression of ClfA-CTA2/B, and (B) SDS-PAGE of purified

IsdA-CTA

2/B (1, IsdA-CTA2~38 kD, CTB~11 kD) and ClfA-CTA2/B (2, ClfA-CTA2~37 kD, CTB~11

kD).

Figure 1.

S. aureus cholera toxin A

/B (CTA

/B) chimeric mucosal vaccines. (

) pLR001 for expression

of IsdA-CTA

/B, and pLR003 for expression of ClfA-CTA

/B, and (

) SDS-PAGE of purified IsdA-

CTA

/B (1, IsdA-CTA

~38 kD, CTB~11 kD) and ClfA-CTA

/B (2, ClfA-CTA

~37 kD, CTB~11 kD).

2.2. Protein Expression and Puriﬁcation

Chimeras were puriﬁed as previously described [

]. Brieﬂy, to express IsdA-

CTA

/B and ClfA-CTA

/B, ClearColi

with pLR001 or pLR003 were grown at 37

◦

to an O.D. of 0.9 at 600 nm and induced for 24 h with 0.2% L-arabinose. Proteins were

isolated from the periplasmic extract with 1 mg/mL polymyxin B and puriﬁed by afﬁnity

Vaccines 2021, 9, 6 4 of 17

chromatography on immobilized D-galactose (Pierce

™

D-Galactose Agarose, Thermo

Fisher, Waltham, MA, USA). Vaccine proteins were dialyzed into sterile 5% glycerol +

1X PBS and concentrations were determined by bicinchoninic acid assay (BCA) (Pierce

™

BCA, Thermo Fisher, Waltham, MA, USA). Sizes and purities of the vaccine chimeras

were conﬁrmed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-

PAGE) prior to mixing at a ﬁnal protein concentration of 600

g/5 mL for vaccination

(Figure 1B). Vaccines were tested to ensure endotoxin levels were below 0.05 EU/mL (LAL

Endpoint Chromogenic, Lonza, Allendale, NJ, USA), plated for sterility on tryptic soy agar,

and stored at −80

◦

C until use.

2.3. Animals, Vaccination, Challenge, and Clinical Assessment

All animal protocols were pre-approved by the University of Idaho Animal Care

and Use Committee. Lactating healthy Holstein cows in the third or fourth lactation

were pre-screened for inclusion as being those with two consecutive SCC readings below

200

cells/mL and no clinical evidence of mastitis. Further enrollment criteria were

followed as described previously [

] and included: (1) no growth of S. aureus culture from

milk as determined by plating on mannitol salt agar (MSA) and PCR with

S. aureus nuc

and isdA primers, (2) low baseline anti-IsdA responses as determined by enzyme-linked

immunosorbent assay (ELISA) of milk and serum, and (3) no evidence of bovine leukemia

virus infection (Washington Animal Disease Diagnostic Lab, WADDL, Pullman, WA, USA).

Seven selected cows were ultimately randomized into vaccinated and control groups.

Figure 2 shows the summary of trial design. Four vaccinated animals received a 600

intranasal dose of IsdA + ClfA-CTA

/B in 1X PBS + 5% glycerol on days 1 and 14 (or-

ange arrows, blue bar), and a control group of three animals of similar age and lactation

period received vehicle control (1X PBS + 5% glycerol) mock vaccination on days 1 and 14

(orange arrows, grey bar). The vaccine was delivered in 2.5 mL volumes into each nare

using a nasal cannula (Merck & Co., Kenilworth, NJ, USA). On day 20, all animals were

challenged in two quarters with 400 CFU in 1 mL of S. aureus Newbould 305 (yellow arrow).

Quarters were identiﬁed as left front (LF), left rear (LR), right front (RF), and right rear (RR).

The bacterial challenge was inoculated into two diagonal quarters of each vaccinated cow

using teat cannulae (Valley Vet Supply, Marysville, KS, USA). Animals were monitored

closely during the challenge period (days 20 to 30) and evaluated for the presence of clinical

mastitis by assessment of rectal temperature (Figure 3), milk quality (Figure S2), and udder

consistency (examination for edema, hardening, and/or swelling, Table S1) on days of

milk sampling during the trial (Figure 2) [

]. Enrolled animals that developed pain

and/or fever that exceeded 103

◦

F were administered painkillers (Banamine

and aspirin)

as recommended by the attending veterinarian. Shortly after challenge (day 21), one vacci-

nated animal developed a severe Escherichia coli mastitis case in an unchallenged quarter

(2779 LF) with systemic illness including septicemia. Thereafter, the other three quarters

were involved and the animal developed clinical mastitis due to

Staphylococcus aureus

The animal was euthanized on day 5 post-challenge. Results from this animal are not

included in the data in this report and resulting sample size was n = 3 per group, as repre-

sented in

Figure 2

. On day 30 all other animals began treatment with Spectramast (Zoetis,

Parsippany, NJ, USA) until consecutive negative cultures were indicative of safe release to

herd as determined by the attending veterinarian.

Vaccines 2021, 9, 6 5 of 17

Vaccines 2021, 9, x 5 of 17

Figure 2. Trial design summary. Animals (n = 3 per group, #) were vaccinated intranasally on day 1 and boosted on day

14 with 5 mL of either phosphate-buffered saline (PBS) + 5% glycerol vehicle control or 600 µg IsdA + ClfA-CTA2/B vaccine

(orange arrows). On day 20 animals were challenged once with 400 colony-forming units (CFU) of S. aureus Newbould

305 in two quarters (yellow arrow) and on day 30, animals were treated (end of challenge period, green arrow). Samples

of blood were taken on days 1, 14, 20, and 30 (X). Samples of milk were taken on days 1 and 14 (X), and every day for ten

days over the challenge period (days 20–30, XX).

Figure 2.

Trial design summary. Animals (n = 3 per group, #) were vaccinated intranasally on day 1 and boosted on day 14

with 5 mL of either phosphate-buffered saline (PBS) + 5% glycerol vehicle control or 600

g IsdA + ClfA-CTA

/B vaccine

(orange arrows). On day 20 animals were challenged once with 400 colony-forming units (CFU) of S. aureus Newbould 305

in two quarters (yellow arrow) and on day 30, animals were treated (end of challenge period, green arrow). Samples of

blood were taken on days 1, 14, 20, and 30 (X). Samples of milk were taken on days 1 and 14 (X), and every day for ten days

over the challenge period (days 20–30, X→X).

2.4. Sample Collection and Milk Culture

Blood and milk were sampled on day

−

2 for screening and then on days 1, 14,

20, and 30, and milk was sampled twice daily during the challenge period (Figure 2).

Blood was collected from the tail vein and allowed to coagulate at room temperature (RT)

for 1 h prior to centrifugation and resuspension into 1:10 inhibitor solution (IS, 1X HALT

™

protease inhibitor and 5% glycerol in 1X PBS). On day 20, whole blood was also collected in

vacutainer tubes for peripheral blood mononuclear cell (PBMC) isolation (Becton Dickinson,

Franklin Lakes, NJ, USA). Milk was collected aseptically as 50 mL quarter samples after

washing teat ends with 70% ethanol and was aliquoted into three equal tubes for culture,

SCC, and ELISA. For SCC, milk was ﬁxed prior to shipping and analysis was performed

using the California Mastitis Test (WADDL, Pullman, WA, USA). For ELISA, milk was

centrifuged at 700

g for 20 min at 4

◦

C to remove fat. Skim milk was collected and

centrifuged at 20,000

g for 30 min at 4

◦

C. Whey was collected and stored in 1:10 IS.

Equal volumes of diluted whey from each quarter were pooled and stored at

−

◦

C prior

to analysis. For milk culture, 100

L and 10

L of tenfold serially-diluted quarter milk

was plated on MSA, BA, and MP2 agar (Udder Health Systems, Inc., Meridian, ID, USA)

to determine the number of colony-forming units per mL (CFU/mL). The presence of

larger yellow colonies with yellow zones on MSA, beta-hemolysis on BA, or small, white,

esculin-negative colonies on MP2 was considered presumptive S. aureus. These colonies

were isolated and conﬁrmed by a positive coagulase test or a PCR test using nuc or isdA

primers [

]. CFU by quarter data, based upon ﬁnal quantitation on MSA, was determined

once daily on days

−

2, 1, 14, and 20 prior to challenge and twice daily (AM/PM) during the

challenge period. Quarter data were combined and total CFU/mL by cow was reported

for six animals (n = 3 per group).

Vaccines 2021, 9, 6 6 of 17

Vaccines 2021, 9, x 6 of 17

Figure 3. Vaccination outcomes during the trial period. (A) Quantification of bacterial shedding by

cows during the challenge period. Log10 of CFU/mL of Staphylococcus aureus on mannitol salt agar

(MSA). Mean ± standard error, n = 3 per group, and analyzed using repeated measures analysis of

variance (ANOVA). No significance after false discovery rate (FDR) adjustment for multiple com-

parisons. (B) Somatic cell count (SCC) (×1000 cells/mL) by cow. Mean ± standard error, n = 3 per

group, and analyzed using repeated measures ANOVA. During the challenge period, control

cows uniformly had higher SCC than vaccinated cows (main model effect p = 0.002). (C) Rectal

temperature in degrees Fahrenheit (°F). Mean ± standard error, n = 3 per group, and analyzed us-

ing repeated measures ANOVA showing no significance between groups. Orange arrows indicate

Figure 3.

Vaccination outcomes during the trial period. (

) Quantiﬁcation of bacterial shedding by

cows during the challenge period. Log10 of CFU/mL of Staphylococcus aureus on mannitol salt agar

(MSA). Mean

standard error, n = 3 per group, and analyzed using repeated measures analysis

of variance (ANOVA). No signiﬁcance after false discovery rate (FDR) adjustment for multiple

comparisons. (

) Somatic cell count (SCC) (

1000 cells/mL) by cow. Mean

standard error, n = 3

per group, and analyzed using repeated measures ANOVA. During the challenge period, control cows

uniformly had higher SCC than vaccinated cows (main model effect p = 0.002). (

) Rectal temperature

in degrees Fahrenheit (

◦

F). Mean

standard error, n = 3 per group, and analyzed using repeated

measures ANOVA showing no signiﬁcance between groups. Orange arrows indicate day of booster

vaccination (14), yellow arrows indicate day of bacterial challenge (20) and green arrows indicates

the last day of challenge (30).

Vaccines 2021, 9, 6 7 of 17

2.5. IgG, IgG1, IgG2, and IgA Enzyme-Linked Immunosorbent Assay (ELISA)

IsdA- and ClfA-speciﬁc immune responses in serum and milk were detected using

ELISA as described [

]. Brieﬂy, 96-well microtiter plates (Nunc, Thermo Fisher, Waltham,

MA, USA) were coated with 10

g of either IsdA-CTA

/B, ClfA-CTA

/B, or CTA

/B in

1X PBS and incubated overnight at 4

◦

C. Coated plates were blocked for 2 h at 37

◦

C in

1% goat milk + 1X PBS. After washing, plates were incubated with two-fold dilutions of

either bovine serum (dilutions initiated at 1:200 concentration) or pooled quarter milk

(dilutions initiated at a 1:10 concentration). Plates were incubated at 4

◦

C overnight.

After washing, plates were incubated with horseradish peroxidase (HRP)-conjugated anti-

bovine IgG, IgG1, IgG2, or IgA (1:10,000 Bethyl Laboratories, Montgomery, TX, USA)

at 37

◦

C for 1 h. Plates were developed with tetramethylbenzidine (Promega

TMB

One, Thermo Fisher, Waltham, MA, USA) and read at 370 nm per TMB manufacturer’s

instruction. ELISA results from serum or pooled quarter milk were reported by cow

(n = 3) and presented as the ratio of results (day X/day 1) of the O.D. (370 nm) from a

representative antibody dilution in the linear part of the curve (1:1600 serum, 1:160 milk).

Results are the average of three independent assays.

2.6. Peripheral Blood Mononuclear Cell (PBMC) Isolation and Cytokine qPCR

PBMCs were isolated from whole bovine blood on day 20 for cytokine analysis. PBMCs

were isolated using a density gradient established by layering whole blood diluted 1:2 with

1X PBS on Histopaque

-1077 (Sigma-Aldrich, St. Louis, MO, USA). Blood samples were cen-

trifuged at 800

g for 30 min at RT. The buffy coat was removed and washed three times by

centrifugation with Hank’s Balanced Salt Solution for 10 min at 400

g at RT, and cells were

counted with 0.2% trypan blue. For cytokine assays, total RNA from PBMCs from each cow

(n = 3 per group) was extracted (RNeasy, Qiagen, Germantown, MD, USA) with an addi-

tional Dnase I (Promega, Madison, WI, USA) digestion. cDNA was reverse transcribed per

manufacturer’s instructions (High-Capacity RNA-to-cDNA

™

Kit, Thermo Fisher, Waltham,

MA, USA). qRT-PCR was conducted using SYBR fast (Kapa Biosystems, Thermo Fisher,

Waltham, MA, USA) on interferon gamma (

IFN-γ

), interleukin-6 (IL-6), and interleukin-

10 (IL-10) primers, using bovine glyceraldehyde

3-phosphate

dehydrogenase (GAPDH)

as a reference gene (primers, Table 1). Results are presented as relative gene expression

−∆∆Ct

[

]. All qRT-PCR experiments were performed in triplicate per cow PBMC sample.

2.7. Sample Size, Statistical Methods, and Analysis

Sample size was estimated prior to study by power analysis based upon predicted

SCC and CFU/mL in milk and using the assumption that quarters are independent,

as has been reported [

]. A sample size of 13 quarters per group was predicted

to provide, at a 95% level of conﬁdence, 80% power to detect a difference in logged

SCC. Resulting quarter bacterial counts and SCC data from this study were analyzed

by (1) assuming independent quarters and (2) as the combined average of quarters by

cow. Outcomes were not different, thus results are reported as the average by cow and

assuming quarters are not independent. The log-base 10 values of CFU, SCC, temperature,

and serum and milk anti-IsdA, ClfA, and CTB antibodies were analyzed using repeated

measures analysis of variance (ANOVA) with time as the within-subjects variable and

group as the between-subjects variable. Within-subjects correlation was modeled with

either ﬁrst-order autoregressive or compound symmetric structure, depending on Akaike’s

Information Criterion [

]. Comparisons of interest were identiﬁed prior to modeling

and were examined regardless of the signiﬁcance of main effects or interaction. First,

we explicitly compared the outcome at each study time point. Second, we examined the

change in outcome within group, comparing days and adjusting the paired comparisons

using false discovery rate [

]. Cytokine analysis was performed using a two-group t-test

between vaccinated and control animals. Statistical analyses were conducted using JMP and

SAS software (Cary, NC). p-values are reported as p

≤

0.05(*), p

≤

0.01(**), or p

≤

0.0001(****)

and reﬂect two-sided tests.

Vaccines 2021, 9, 6 8 of 17

3. Results

3.1. Bacterial Culture and Clinical Assessment

Quantiﬁcation of S. aureus was determined after plating milk that had been sampled

once daily on days

−

2, 1, 14, and 20 prior to challenge and twice daily during the challenge

period (Figure 2). Prior to challenge no animals were found to be shedding S. aureus,

and immediately after challenge all animals shed high levels of S. aureus from infected

quarters (Figure 3A). Results revealed a rapid decline in bacterial shedding from all animals

within 24 h and then a slow decline beginning in the middle of the challenge period.

Between days 2 and 10 of the challenge period (days 22 and 30 of trial) control animals

shed a total of 1.08

CFU/mL and vaccinated animals shed 7.53

CFU/mL.

Differences between treatment groups were observed on days 21, 29, and 30 during the

challenge period (p = 0.029, 0.011, and 0.018, respectively), however, after adjusting for

multiple comparisons, these results are not signiﬁcant. S. aureus was isolated from all

challenged quarters in both treatment groups, and all animals continued to shed S. aureus

throughout the trial. Animals did not shed from uninfected quarters. While one vaccinated

animal was culture negative at two time points late in the challenge period (day 29 AM and

day 30 AM), no animals were consistently sterile of S. aureus by the end of the challenge

period. Analysis of positive quarters indicated that there were more days showing a lower

percentage of infected quarters for vaccinated animals (Supplementary Figure S1).

SCC taken once daily before the challenge period and twice daily during challenge

is shown in Figure 3B. Results show a consistently reduced SCC from vaccinated ani-

mals beginning 48 h post-challenge (day 22). While individual days were not signiﬁcant

after adjustment, across and after the challenge period (days 21 to 39) unvaccinated an-

imals had signiﬁcantly-higher SCC than vaccinated animals (model main effect of treat-

ment group, p = 0.002). SCCs of individual cows throughout the trial are shown in

Supplementary Figure S3B.

The average rectal temperature per group during the challenge period is shown

Figure 3C

. Temperatures at 72 h post-challenge (day 23 AM) showed an average of

102.7

◦

F for control and 101.5

◦

F for vaccinated animals, however there was no statistically-

signiﬁcant difference in temperature between groups on any day during this period. In ad-

dition, no differences in temperature between groups occurred within 24 h after vaccination

(days 1 and 14).

Clinical assessments indicated that animals did not show signs of systemic illness,

loss of appetite, or adverse local reactions due to the vaccine, and no animals had clinical

evidence of mastitis prior to challenge (day 20). Clinical results are summarized in Supple-

mentary Table S1. Clinical mastitis due to S. aureus was observed in challenged quarters of

control cows 2767 (LF and RF) and 2830 (LR) throughout the evaluation period. The latter

cow developed a persistent mastitis starting on day 23 with apparent milk changes that

included clots and ﬂakes in the LR quarter. Clinical mastitis in this animal included persis-

tent udder swelling in addition to pain, heat, and sensation of the affected teat until the end

of the challenge period. Temporary enlargement of the supramammary lymph node was

noted in one of the vaccinated cows (2823) on day 24, and persistent enlargement observed

in one control animal (2830).

Milk quality assessments indicated that while the fat, protein, lactose, and solids-not-

fat (SNF) percentages were frequently higher in vaccinated animals, these differences were

not statistically signiﬁcant (Supplementary Figure S2).

3.2. Vaccine-Speciﬁc Antibody Responses in Blood and Milk

Antigen-specific humoral responses were quantified by ELISA from blood and milk.

Anti-IgG responses in serum on days 14, 20 and 30, relative to day 1, are shown in

Figure 4A,C and E

. Vaccinated animals (blue bars) showed a significant IsdA-CTA

/B-

specific IgG response in serum after challenge on day 30 relative to days 14 and

20 (p

adj

= 0.008

for both) and on day 30 relative to control animals (p = 0.030 *) (

Figure 4A

). Vaccinated ani-

mals showed a similar, but non-signiﬁcant, anti-ClfA-CTA

/B-speciﬁc IgG responses in

Vaccines 2021, 9, 6 9 of 17

serum on day 30 relative to days 14 and 20 (p

adj

= 0.120) as well as on day 30 relative

to control animals (p = 0.079) (Figure 4C). Anti-CTA

/B-speciﬁc IgG responses in serum

remained low and non-signiﬁcant between groups throughout and after challenge (day

30) (Figure 4E).

Vaccines 2021, 9, x 10 of 17

Figure 4. Immunoglobulin G (IgG) antibody responses in serum and milk as determined by en-

zyme-linked immunosorbent assay (ELISA). Anti-IsdA-CTA

2/B IgG responses in (A) serum and

(B) milk, anti-ClfA-CTA2/B IgG responses in (C) serum and (D) milk, and anti-CTA2/B IgG re-

sponses in (E) serum and (F) milk. Serum was analyzed on days 14, 20, and 30 and milk on days

14, 20, 22, 24, 26, 28, and 30 during the trial period. Results are reported as ELISA ratios of day

X/day 1 at O.D. 370 at serum dilutions of 1:1600 and milk dilutions of 1:160. Shown are mean and

standard error by treatment with control (gray) and vaccinated (blue) (n = 3 per group). Significant

differences between groups are represented as p ≤ 0.05 (*). The log10 of the values were analyzed

using repeated measures analysis of variance (ANOVA) with a compound symmetric covariance

structure for cows across days. Model-based estimates were compared between groups within

days and adjusted for multiple comparisons.

Anti-IgG responses in milk on days 14, 22, 24, 26, 28, and 30, relative to day 1, are

shown in Figure 4D–F. IsdA-CTA

2/B-specific IgG responses in milk increased over the

challenge period in vaccinated animals, with values significantly higher on day 30 relative

to days 20 to 26 (adjusted p-values all <0.05) and on day 30 relative to control cows (p =

0.030 *) (Figure 4D). The anti-IsdA-CTA

2/B differences between days for control cows

were non-significant after day 20. Anti-ClfA-CTA

2/B-specific IgG responses in milk were

significant on day 30 relative to days 20–24 (adjusted p-values all <0.05) for the vaccinated

group and on day 30 relative to unvaccinated cows (p = 0.043 *) (Figure 4E). Milk anti-

CTA

2/B-specific IgG responses increased moderately during the challenge period in both

vaccinated and control animals with significant increases on day 30 relative to days 14

Figure 4.

Immunoglobulin G (IgG) antibody responses in serum and milk as determined by enzyme-

linked immunosorbent assay (ELISA). Anti-IsdA-CTA

/B IgG responses in (

) serum and (

) milk,

anti-ClfA-CTA

/B IgG responses in (

) serum and (

) milk, and anti-CTA

/B IgG responses in (

)

serum and (

) milk. Serum was analyzed on days 14, 20, and 30 and milk on days 14, 20, 22, 24,

26, 28, and 30 during the trial period. Results are reported as ELISA ratios of day X/day 1 at O.D.

370 at serum dilutions of 1:1600 and milk dilutions of 1:160. Shown are mean and standard error

by treatment with control (gray) and vaccinated (blue) (n = 3 per group). Signiﬁcant differences

between groups are represented as p

≤

0.05 (*). The log10 of the values were analyzed using repeated

measures analysis of variance (ANOVA) with a compound symmetric covariance structure for cows

across days. Model-based estimates were compared between groups within days and adjusted for

multiple comparisons.

Anti-IgG responses in milk on days 14, 22, 24, 26, 28, and 30, relative to day 1,

are shown in Figure 4B, D and F. IsdA-CTA

/B-speciﬁc IgG responses in milk increased

over the challenge period in vaccinated animals, with values signiﬁcantly higher on day 30

relative to days 20 to 26 (adjusted p-values all <0.05) and on day 30 relative to control cows

(p = 0.030 *) (Figure 4B). The anti-IsdA-CTA

/B differences between days for control cows

Vaccines 2021, 9, 6 10 of 17

were non-signiﬁcant after day 20. Anti-ClfA-CTA

/B-speciﬁc IgG responses in milk were

signiﬁcant on day 30 relative to days 20–24 (adjusted p-values all <0.05) for the vaccinated

group and on day 30 relative to unvaccinated cows (p = 0.043 *) (Figure 4D). Milk anti-

CTA

/B-speciﬁc IgG responses increased moderately during the challenge period in both

vaccinated and control animals with signiﬁcant increases on day 30 relative to days 14

and 20 in the vaccinated group and no change in the control group (adjusted p-values all

<0.05). The differences in anti-CTA

B responses between vaccine and control groups were

non-signiﬁcant on all days tested (Figure 4F).

Serum IgG subtype (IgG1 and IgG2) responses were evaluated to further deﬁne

the T helper immune response (Figure 5A–D). Vaccinated animals exhibited increases

in IgG1 and IgG2 responses on day 30 for both the IsdA- and ClfA-CTA

/B antigens.

The serum anti-IsdA-CTA

/B IgG1 response on day 30 relative to days 14 and 20 was

signiﬁcant for vaccinated animals (blue bars, p

adj

= 0.004 and p

adj

= 0.007, respectively),

and the difference between groups was signiﬁcant on day 30 (p = 0.033 *) (Figure 5A).

For anti-IsdA-CTA

/B IgG2 responses, day 30 was higher than days 14 and 20 for both

vaccinated and control groups (p

adj

= 0.045 for both comparisons) with no signiﬁcant

differences between groups on day 30 (Figure 5B). For serum anti-ClfA-CTA

/B IgG1,

vaccinated animals showed an increase on day 30 compared to day 14 (p

adj

= 0.023) and day

20 (p

adj

= 0.029), and the difference between groups was signiﬁcant on day 30 (p = 0.029 *)

(

Figure 5C

). For anti-ClfA-CTA

/B IgG2 responses, day 30 was higher than days 14 and 20

for both groups as well (p

adj

= 0.015 for both comparisons), however the difference between

vaccinated and control groups on day 30 was non-signiﬁcant after adjustment (p = 0.050)

(

Figure 5D

). Assessment of milk anti-IsdA-CTA

/B and anti-ClfA-CTA

/B IgG1, IgG2 and

IgA responses was also performed, and while results indicated an increase on day 30 for

both IgG1 and IgA, they were non-signiﬁcant between vaccine and control groups on the

days (14, 20, and 30) tested (data not shown).

Combined, ELISA analysis shows an induction of antigen-speciﬁc humoral responses

in the milk and serum after intranasal IsdA + ClfA-CTA

/B vaccination, as evidenced by

a signiﬁcant booster effect upon bacterial challenge. Antibody subtyping indicated that

both antigens stimulated a Th2-type response, with ClfA potentially inducing a mixed

Th1/Th2 response. Lastly, there was no signiﬁcant antibody response to the CTA

adjuvant vector alone.

3.3. Cytokine Assay

The stimulation of cellular cytokine responses was assessed by quantitative RT-PCR

using PBMCs isolated from vaccinated and control cows on day 20 (Figure 5E). IL-12,

TNF-

, and IL-4 levels were not signiﬁcantly different between vaccinated and control

animals (data not shown). Vaccinated cows showed a slight but signiﬁcant increase in

IFN-

expression (p = 0.048 *) but no signiﬁcant difference in IL-10 or IL-6 expression

(Figure 5E).

Vaccines 2021, 9, 6 11 of 17

Vaccines 2021, 9, x 11 of 17

and 20 in the vaccinated group and no change in the control group (adjusted p-values all

<0.05). The differences in anti-CTA

2B responses between vaccine and control groups were

non-significant on all days tested (Figure 4F).

Serum IgG subtype (IgG1 and IgG2) responses were evaluated to further define the

T helper immune response (Figure 5A–D). Vaccinated animals exhibited increases in IgG1

and IgG2 responses on day 30 for both the IsdA- and ClfA-CTA

2/B antigens. The serum

anti-IsdA-CTA

2/B IgG1 response on day 30 relative to days 14 and 20 was significant for

vaccinated animals (blue bars, p

adj = 0.004 and padj = 0.007, respectively), and the difference

between groups was significant on day 30 (p = 0.033 *) (Figure 5A). For anti-IsdA-CTA

2/B

IgG2 responses, day 30 was higher than days 14 and 20 for both vaccinated and control

groups (p

adj = 0.045 for both comparisons) with no significant differences between groups

on day 30 (Figure 5B). For serum anti-ClfA-CTA

2/B IgG1, vaccinated animals showed an

increase on day 30 compared to day 14 (p

adj = 0.023) and day 20 (padj = 0.029), and the dif-

ference between groups was significant on day 30 (p = 0.029 *) (Figure 5C). For anti-ClfA-

CTA

2/B IgG2 responses, day 30 was higher than days 14 and 20 for both groups as well

adj = 0.015 for both comparisons), however the difference between vaccinated and control

groups on day 30 was non-significant after adjustment (p = 0.050) (Figure 5D). Assessment

of milk anti-IsdA-CTA

2/B and anti-ClfA-CTA2/B IgG1, IgG2 and IgA responses was also

performed, and while results indicated an increase on day 30 for both IgG1 and IgA, they

were non-significant between vaccine and control groups on the days (14, 20, and 30)

tested (data not shown).

Figure 5. Serum IgG1, IgG2, and cytokine expression analysis. (A) Anti-IsdA-CTA2/B IgG1, (B)

anti-IsdA-CTA2/B IgG2, (C) anti-ClfA-CTA2/B IgG1, and (D) anti-ClfA-CTA2/B IgG2 responses in

serum on days 14, 20, and 30. Results are reported as ELISA ratios of day X/day 1 at O.D. 370 at

serum dilutions of 1:1600. Shown are mean and standard error by treatment with control (gray)

and vaccinated (blue) (n = 3 per group). The log10 of the values were analyzed using analysis of

variance (ANOVA), with a compound symmetric covariance structure for cows across days.

Model-based estimates were compared between groups within days and adjusted for multiple

comparisons. (E) IL-10, IL-6, and IFN-γ expression as determined by quantitative RT-PCR of pe-

ripheral blood mononuclear cells (PBMCs) isolated from whole blood after boost on day 20. Re-

sults are shown as relative gene expression to GAPDH (2

−ΔΔCt

). Data are presented as mean and

standard error of control (gray) and vaccinated (blue) showing median and range (n = 3 per

Figure 5.

Serum IgG1, IgG2, and cytokine expression analysis. (

) Anti-IsdA-CTA

/B IgG1, (

) anti-IsdA-CTA

/B IgG2,

(

) anti-ClfA-CTA

/B IgG1, and (

) anti-ClfA-CTA

/B IgG2 responses in serum on days 14, 20, and 30. Results are reported

as ELISA ratios of day X/day 1 at O.D. 370 at serum dilutions of 1:1600. Shown are mean and standard error by treatment

with control (gray) and vaccinated (blue) (n = 3 per group). The log10 of the values were analyzed using analysis of variance

(ANOVA), with a compound symmetric covariance structure for cows across days. Model-based estimates were compared

between groups within days and adjusted for multiple comparisons. (

) IL-10, IL-6, and IFN-

expression as determined

by quantitative RT-PCR of peripheral blood mononuclear cells (PBMCs) isolated from whole blood after boost on day 20.

Results are shown as relative gene expression to GAPDH (2

−∆∆Ct

). Data are presented as mean and standard error of

control (gray) and vaccinated (blue) showing median and range (n = 3 per group). Data were analyzed using a two-group

t-test between vaccinated and control. Signiﬁcant differences between groups are represented as p ≤ 0.05 (*).

4. Discussion

This report describes the outcomes of a small bovine challenge trial to assess the efﬁ-

cacy of the IsdA + ClfA-CTA

/B mucosal S. aureus mastitis vaccine. We hypothesized that

vaccination would prevent or reduce bacterial shedding from the udder after intramam-

mary challenge and reduce disease outcomes. Animals were vaccinated intranasally during

milking and challenged in two quarters with the homologous S. aureus Newbould 305

vaccine strain. An averaged reduction in CFU/mL from combined quarters of vaccinated

compared to unvaccinated animals was observed beginning 24 h after challenge to the end

of the challenge period, however, this difference was not signiﬁcant on speciﬁc days during

the challenge period. Analysis of bacteriology using independent quarters did not change

data interpretations, however, a lower percentage of infected quarters was observed on

multiple days after challenge. Analysis of SCC revealed that vaccinated animals had lower

numbers of cells on the majority of days during the challenge period of the trial, and this

decrease was signiﬁcant between vaccinated and control animals during the whole of the

period. These results were also consistent with the evidence of reduced clinical mastitis in

vaccinated animals.

The assessment of humoral immune responses in milk and serum in this report showed

induction of IsdA- and ClfA-CTA

/B speciﬁc IgG antibodies in vaccinated animals after

S. aureus

challenge indicating that vaccination induced antigen-speciﬁc responses that were

ampliﬁed by bacterial challenge. In contrast to previous studies, no signiﬁcant increase in

Vaccines 2021, 9, 6 12 of 17

antigen-speciﬁc humoral responses was detected in the serum directly after vaccination

and boost, despite the same vaccine dose and schedule [

].The lower sample size in this

trial compared to previous trials with IsdA + ClfA-CTA

/B may have contributed to this

outcome, and larger trials will be essential to advance this vaccine candidate. In addition,

animals were vaccinated during milking for this study instead of during dry-off, which is

a period of higher susceptibility to mastitis and changes in immune function that may

explain observed differences in immunogenicity. As with previous trials, antibody analysis

revealed that not all vaccinated animals responded well to the same vaccine preparation

and dosage. Variations in host genetics or inconsistencies in administration can cause

these disparities, and larger trials will help to exclude them. Other vaccination routes,

or alternate prime-boost strategies, may also promote vaccination consistency and efﬁcacy.

These routes were not explored in this early study to enable a narrow focus on mucosal

delivery, but intramuscular, subcutaneous, and transdermal routes are all effective for

CT-adjuvanted vaccines and could be explored. Lastly, in this study we maintained a short

dosage interval of only 14 days to align with previous trials, however, a longer interval

between doses may improve responses and will be explored in the future.

Animals were vaccinated during milking to permit bacterial quantiﬁcation and limit

the potential for systemic or chronic infection. Despite this, one vaccinated animal was

euthanized shortly after challenge due to an E. coli infection that rapidly became systemic.

While little has been reported about the effects of co-infection on the severity of E. coli

mastitis, the cow immune status is a key factor, and S. aureus is known for the production

of virulence factors that modulate the immune response [

]. Speciﬁcally the

S. aureus

superantigens (SAgs) can activate speciﬁc T-cell subsets, resulting in inﬂammation, tis-

sue damage, and potential T-cell anergy [

–

]. S. aureus Newbould 305 strain was

chosen for these studies because it induces mild and chronic mastitis, has been utilized

before in vaccine challenge trials, and contains a limited repertoire of SAgs [

]. It is

recognized, however, that immune dysregulation likely occurred upon challenge and,

despite vaccination, contributed to the enhanced spread and systemic infection in this

animal. The potential for co-infection and the ability of the vaccine to protect against

heterologous S. aureus isolates that may induce more severe disease will both need to be

addressed in future studies.

As described above, CT and its non-toxic B subunit can induce humoral and cellu-

lar immune responses to co-delivered antigens. CTA

/B molecules retain much of the

well-characterized adjuvanticity of CTB to induce both humoral and cellular responses.

The IgG1 and IgG2 proﬁles we observed in the serum of vaccinated animals on day 30 were

consistent with our previous studies indicating that CTA

/B chimeras promote a largely

Th2-type cellular response [

]. In the current study, however, the responses to IsdA

were more clearly polarized toward Th2, while the anti-ClfA responses are supportive of

a potential mixed Th1/Th2 response. Cytokine expression analysis in the current study,

performed on day 20 prior to challenge, showed no effect on IL-10 and IL-6, but an increase

in IFN-

in vaccinated animals. Cytokine analysis from previous immunogenicity studies

using the IsdA + ClfA-CTA

/B vaccine largely supported a Th2-type response and did not

indicate IFN-

upregulation [

]. This apparent contradiction may be due to differences

in the timing of analysis (6 days after vaccination in the current study versus 45 days after

vaccination in previous studies) and the methods used (unstimulated versus stimulated

PBMCs). Reports indicate that while CTB more commonly induces Th2-type responses,

it can induce a mixed Th2/Th1 response with enhanced IFN-

secretion, depending upon

the antigen and route of delivery [

–

]. Similar to CTB, vaccination with CTA

chimeras may promote early macrophage or dendritic cell activation and antigen presen-

tation through IFN-

upregulation. In this study there was not a clear early effect on the

inﬂammatory and pro-inﬂammatory balance of serum IL-6 and IL-10, however, others have

reported anti-inﬂammatory properties in CT and its derivatives. These properties may be

advantageous for the prevention of S. aureus udder colonization and are consistent with

our observed reduction in SCC after challenge [72–74].

Vaccines 2021, 9, 6 13 of 17

Lastly, in this study we determined if animals responded to the vaccine adjuvant alone

by producing anti-CTA

/B humoral responses. Results showed no signiﬁcant differences

between vaccinated and control groups. While S. aureus challenge was not expected

to induce anti-CT antibodies, previous studies have reported the undesirable effect of

signiﬁcant anti-CT antibodies after use of this adjuvant for mucosal vaccination [

The low adjuvant-speciﬁc antibody response observed here, combined with the reduced

recruitment of somatic cells, provides support for the utility of CTA

/B-based vaccines.

These studies indicate that IsdA + ClfA-CTA

/B may be effective in the reduction

S. aureus

colonization and clinical outcome, as evidenced by reduced SCC, but do not

provide evidence of complete protection or elimination. Both vaccinated and unvacci-

nated animals shed high levels of S. aureus Newbould 305 immediately after challenge,

and all animals in the study were found to shed the challenge strain during the entire

10-day

challenge period. This outcome may be the result of a high bacterial dose and

the artiﬁcial nature of intramammary challenge. The use of a lower challenge dose, a dif-

ferent method of challenge, and/or focus on natural transmission in a larger ﬁeld trial

will better determine efﬁcacy to prevent infection. In addition, studies are needed that

utilize heterologous isolates, compare outcomes with current vaccines, and assess alternate

routes of immunization. IsdA and ClfA are established and highly-conserved antigens

from bovine S. aureus, however, the incorporation of additional antigens, including toxins

and anti-immune factors, may also be necessary to promote strain cross-protection and

control immune modulation.

5. Conclusions

Results indicate vaccine efﬁcacy in reducing SCC and improving clinical outcome and

support further exploration of the IsdA + ClfA-CTA

/B vaccine to prevent bovine mastitis.

The development of an effective vaccine to prevent mastitis caused by S. aureus would have

many positive impacts on animal health and food production and may decrease overall

antibiotic use in the industry. Needle-free vaccination of cattle would also be beneﬁcial by

reducing the transmission of disease, inducing mucosal immunity, and promoting vaccine

distribution and use. This study provides important preliminary results of a cholera-

toxin-based intranasal vaccine in a mastitis challenge model and supports the continued

exploration of this antigen-adjuvant platform to prevent bovine disease.

Supplementary Materials:

The following are available online at https://www.mdpi.com/2076-3

93X/9/1/6/s1, Figure S1: Percent S. aureus Newbould 305 infected quarters from vaccinated and

control groups during the challenge period, Figure S2: Milk quality assessment during the trial period,

Figure S3: Individual cow bacterial shedding and SCC during trial, Table S1: Clinical outcomes after

challenge on day 20.

Author Contributions:

All authors were blinded throughout the trial with the exception of J.K.T.

H.A.A. performed trial coordination at the University of Idaho as well as vaccination, sampling,

and cow monitoring. D.S. performed whole-blood PBMC extraction and cytokine analysis as well

as milk DNA qPCR. E.O. performed milk and serum ELISA. J.E.W. performed sample processing,

trial coordination, and shipments. O.M.M. puriﬁed and quality tested vaccines. G.D. processed milk

and blood samples and performed colony counts at Boise State University. L.B. aided with study

design and post-trial statistical analysis. M.A.M. was co-lead investigator at the University of Idaho.

J.K.T. was co-lead investigator at Boise State. All authors have read and agreed to the published

version of the manuscript.

Funding:

This research was funded by a 2013 USDA AFRI standard grant (#2013-01189, PI-Tinker,

Co-PI McGuire

), and a faculty seed grant to J.K.T. from an Institutional Development Award (IDeA)

from the National Institute of General Medical Sciences of the National Institutes of Health

(#P20GM103408 and P20GM109095). We also acknowledge support from The Biomolecular Research

Center at Boise State with funding from the National Science Foundation, Grants # 0619793 and

#0923535, the MJ Murdock Charitable Trust, and the Idaho State Board of Education.

Vaccines 2021, 9, 6 14 of 17

Acknowledgments:

We would like to thank Neha Misra, Brian Mitchell, Laura Rogers, Jim Schroeder,

Edgar Ayala Tapia, and Aurora Thomson-Vogel, for technical support and discussion, as well as

Michael Jobling and Randall Holmes for kind donation of the pARLDR19 vector.

Conﬂicts of Interest:

J.K.T. holds an unlicensed patent for the use of cholera toxin chimera as a

staphylococcal vaccine (Tinker, U.S. Pat. No. 8,834,898).

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