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Comparative Study
. 2013 Jul 23;8(7):e68895.
doi: 10.1371/journal.pone.0068895. Print 2013.

An empirical approach towards the efficient and optimal production of influenza-neutralizing ovine polyclonal antibodies demonstrates that the novel adjuvant CoVaccine HT™ is functionally superior to Freund's adjuvant

Natalie E Stevens  1 , Cara K Fraser, Mohammed Alsharifi, Michael P Brown, Kerrilyn R Diener, John D Hayball
Affiliations
Comparative Study

An empirical approach towards the efficient and optimal production of influenza-neutralizing ovine polyclonal antibodies demonstrates that the novel adjuvant CoVaccine HT™ is functionally superior to Freund's adjuvant

Natalie E Stevens et al. PLoS One. .

Abstract

Passive immunotherapies utilising polyclonal antibodies could have a valuable role in preventing and treating infectious diseases such as influenza, particularly in pandemic situations but also in immunocompromised populations such as the elderly, the chronically immunosuppressed, pregnant women, infants and those with chronic diseases. The aim of this study was to optimise current methods used to generate ovine polyclonal antibodies. Polyclonal antibodies to baculovirus-expressed recombinant influenza haemagglutinin from A/Puerto Rico/8/1934 H1N1 (PR8) were elicited in sheep using various immunisation regimens designed to investigate the priming immunisation route, adjuvant formulation, sheep age, and antigen dose, and to empirically ascertain which combination maximised antibody output. The novel adjuvant CoVaccine HT™ was compared to Freund's adjuvant which is currently the adjuvant of choice for commercial production of ovine polyclonal Fab therapies. CoVaccine HT™ induced significantly higher titres of functional ovine anti-haemagglutinin IgG than Freund's adjuvant but with fewer side effects, including reduced site reactions. Polyclonal hyperimmune sheep sera effectively neutralised influenza virus in vitro and, when given before or after influenza virus challenge, prevented the death of infected mice. Neither the age of the sheep nor the route of antigen administration appeared to influence antibody titre. Moreover, reducing the administrated dose of haemagglutinin antigen minimally affected antibody titre. Together, these results suggest a cost effective way of producing high and sustained yields of functional ovine polyclonal antibodies specifically for the prevention and treatment of globally significant diseases.

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Conflict of interest statement

Competing Interests: JDH undertook a paid scientific consultancy with BTG Australasia Pty Ltd in 2008. This support from BTG Australasia Pty Ltd does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Different routes of prime immunisation yield similar anti-HA antibody titres.
Sheep (n = 5) were immunised with 200 µg rHA SC or IP in complete FA. Sheep were boosted SC every two weeks to a total of five boosts in incomplete FA (indicated by arrows). Pre-immune (time 0) or hyperimmune serum samples were analysed for anti-HA IgG via ELISA (1/50, 000 dilution) (A), and HAI(B). Data are expressed as the mean ± SEM. Two-way repeated-measures ANOVA was applied to evaluate significance which is denoted as thus: * = P<0.05, ** = P<0.01, *** = P<0.001, ns = not significant.
Figure 2
Figure 2. CoVaccine HT™ adjuvant elicits significantly higher anti-HA antibody titres than Freund’s adjuvant.
Sheep (n = 5) were immunised SC with rHA (200 µg) in complete FA or CoVaccine HT™ (CV). Sheep were boosted similarly every two weeks (five boosts indicated by arrows) with rHA in incomplete FA or CV. Pre-immune (time 0) or hyperimmune serum samples were analysed for anti-rHA IgG via ELISA (1/50, 000 dilution) (A) and HAI (B). Data are represented as the mean ± SEM. Two-way repeated-measures ANOVA with Bonferroni post-test was applied to evaluate significance which is denoted as thus: * = P<0.05, ** = P<0.01, *** = P<0.001, ns = not significant.
Figure 3
Figure 3. Nine-month old and three-year old sheep produce similar anti-HA antibody titres.
Sheep (n = 5) at either nine months (young) or three years (old) were immunised SC with 200 µg of rHA in complete FA (A) or CV (B). Sheep were subsequently boosted SC every two weeks to a total of five boosts in incomplete FA or CV (indicated by arrows). Pre-immune (time 0) or hyperimmune serum samples were analysed for anti-rHA IgG via ELISA (1/50, 000 dilution) (Ai, Bi) and HAI (Aii, Bii). Data are expressed as the mean ± SEM. Two-way repeated-measures ANOVA with Bonferroni post-test was applied to evaluate significance; ns = not significant.
Figure 4
Figure 4. Low antigen dose produces similar anti-HA antibody titres.
Sheep (n = 5) were immunised SC with 200 or 20 µg of rHA in complete FA (A) or CV (B). Sheep were then boosted SC every two weeks to a total of five boosts in incomplete FA or CV (indicated by arrows). Pre-immune (time 0) or hyperimmune serum samples at a 1/50,000 dilution were analysed for anti-HA IgG via ELISA (Ai, Bi) and HAI (Aii, Bii). Data are represented as the mean ± SEM endpoint dilution. Data of both assays were analysed by two-way repeated-measures ANOVA with Bonferroni post-tests; significance is denoted as thus: * = P<0.05, ** = P<0.01, *** = P<0.001, ns = not significant.
Figure 5
Figure 5. Repeated subcutaneous immunisation with CoVaccine HT™ elicits fewer reactive immunisation sites than Freund’s adjuvant.
Sheep (n = 15) were immunised SC with rHA antigen either in complete/incomplete FA or CV. Two weeks following the final boost immunisation, injection sites were examined and palpable lumps were enumerated (A) and graded (B). A scoring system was devised based on the size and characteristics of the reaction sites to rank the level of reactivity of each individual site. Grades of site reactivity: 0– no reaction; 1– slight skin irregularity; 2– lump<10 mm diameter or larger skin irregularity; 3– multiple small lumps/single lump<40 mm; 4– lump<80 mm; 5–≥80 mm lump. The data was analysed by Mann-Whitney rank test; significance is denoted as thus: * = P<0.05, ** = P<0.01, *** = P<0.001, ns = not significant.
Figure 6
Figure 6. Prophylactic or therapeutic administration of ovine anti-HA serum is protective against lethal influenza challenge.
Mice (n = 5) were prophylactically administered pooled serum (1 ml, IP) from either young sheep receiving 200 µg rHA SC in CV (Ai) or FA (Aii), day zero pre-bleeds from corresponding sheep from both groups (Aiii) or PBS as a control (Aiv). Twenty-four hours later mice were challenged with a lethal dose of PR8 (500 TCID50). Mice reaching a predetermined endpoint of 20% weight loss (dotted line) were euthanased as indicated by arrows. Mice (n = 5) were challenged with 500 TCID50 PR8 and twenty-four hours later therapeutically administered serum or PBS control as above (Bi–iv). In each panel, data show percentage weight loss of individual mice. Survival curves of mice are also shown (Av, Bv). Mantel-Cox survival analysis was performed on survival curves; significance between all curves is denoted as thus: * = P<0.05, ** = P<0.01, *** = P<0.001, ns = not significant.
Figure 7
Figure 7. Ovine anti-HA serum elicited with CoVaccine HT™ adjuvant show greater in vivo potency as compared to that by Freund’s adjuvant.
Mice (n = 6) were challenged with 500 TCID50 PR8 and twenty-four hours later therapeutically administered dose equivalents of 1000 (A), 500 (B), 250 (C) or 50 µl (D) of pooled hyperimmune serum (1 ml, IP) from sheep that received rHA antigen in CV (i) or FA (ii). Control groups of mice (n = 6) received non-immune serum (Ei) or PBS (Eii) twenty-four hours following viral challenge. Mice reaching a predetermined endpoint of 20% weight loss (dotted line) were euthanased as indicated by arrows. In each panel, data show percentage weight loss of individual mice. Survival curves of mice are also shown (iii). Mantel-Cox survival analysis was performed on survival curves; significance between survival curves is denoted as thus: * = P<0.05, ** = P<0.01, *** = P<0.001, ns = not significant.
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This project was supported in part by funding from the ‘Researchers-in-Business’ scheme (Enterprise Connect, Aust Fed Gov) (to JDH and BTG Australasia Pty Ltd) and the University of South Australia (Div Health Sciences) (to JDH). We also acknowledge the substantive technical, research administrative and material support of BTG Australasia Pty Ltd (Rosedale, South Australia). Accordingly BTG Australasia Pty Ltd (Rosedale, South Australia) had some role in study design and data collection but no role in the analysis, decision to publish, or preparation of the manuscript.