Recombinant herpesvirus glycoprotein G improves the protective immune response to Helicobacter pylori vaccination in a mouse model of disease.

Alphaherpesviruses, which have co-evolved with their hosts for more than 200 million years, evade and subvert host immune responses, in part, by expression of immuno-modulatory molecules. Alphaherpesviruses express a single, broadly conserved chemokine decoy receptor, glycoprotein G (gG), which can bind multiple chemokine classes from multiple species, including human and mouse. Previously, we demonstrated that infection of chickens with an infectious laryngotracheitis virus (ILTV) mutant deficient in gG resulted in altered host immune responses compared to infection with wild-type virus. The ability of gG to disrupt the chemokine network has the potential to be used therapeutically. Here we investigated whether gG from ILTV or equine herpesvirus 1 (EHV-1) could modulate the protective immune response induced by the Helicobacter pylori vaccine antigen, catalase (KatA). Subcutaneous immunisation of mice with KatA together with EHV-1 gG, but not ILTV gG, induced significantly higher anti-KatA IgG than KatA alone. Importantly, subcutaneous or intranasal immunisation with KatA and EHV-1 gG both resulted in significantly lower colonization levels of H. pylori colonization following challenge, compared to mice vaccinated with KatA alone. Indeed, the lowest colonization levels were observed in mice vaccinated with KatA and EHV-1 gG, subcutaneously. In contrast, formulations containing ILTV gG did not affect H. pylori colonisation levels. The difference in efficacy between EHV-1 gG and ILTV gG may reflect the different spectrum of chemokines bound by the two proteins. Together, these data indicate that the immuno-modulatory properties of viral gGs could be harnessed for improving immune responses to vaccine antigens. Future studies should focus on the mechanism of action and whether gG may have other therapeutic applications

Chemokine-binding profiles of EHV-1 gG.

<p>Baculovirus expressed and 6xHis-tag purified EHV-1 gG was incubated with either human, mouse or equine chemokine- or cytokine-coated ELISA wells, before detection with EHV-1 gG-specific antibody. Human IL-8 is used as a positive control for gG binding. Absorbances were measured at 450 nm and backgrounds were subtracted (absorbance in wells containing no gG).</p

Immunisation with KatA and EHV-1 gG, but not ILTV gG, resulted in reduced <i>H. pylori</i> colonisation levels.

<p>BALB/c mice were not vaccinated (unvacc) or vaccinated via the i.n. route recombinant catalase (KatA) alone (10 µg), KatA + CT (10 µg of each) or KatA and either A) EHV-1 gG or B) ILTV gG (10 µg of each). Mice vaccinated subcutaneously received recombinant catalase (KatA) alone (10 µg), formalin-fixed <i>H. pylori</i> (FF-HP) or KatA and either A) EHV-1 gG or B) ILTV gG (10 µg of each), or were not vaccinated (unvacc). Four weeks after the second vaccination, mice were challenged with <i>H. pylori</i> (10⁷, oro-gastric delivery). Box plots represent the median (horizontal line), interquartile range (box) and the 10^th and 90^th percentiles (whiskers). *p<0.05, **p<0.005, ***p<0.0005, compared to unvaccinated group; Kruskal-Wallis with Dunn’s Multiple Comparison test.</p

Immunisation with KatA and EHV-1 gG, but not ILTV gG, boosts specific antibody (IgG) production.

<p>BALB/c mice were remained unvaccinated (unvacc), or were vaccinated via the intranasal or subcutaneous route with either recombinant catalase (KatA) alone (10 µg), KatA (10 µg) adjuvanted with cholera toxin (CT, 10 µg, i.n. only), formalin-fixed <i>H. pylori</i> (FF-Hp, s.c. only) or KatA (10 µg) in combination with either A) EHV-1 gG (10 µg) or B) ILTV gG (10 µg). Two weeks after the second vaccination, tail bleeds were performed and anti-catalase IgG levels in serum were determined. Each point represents an individual mouse; the line represents the median. *p<0.05, ***p<0.0005, ****p<0.00005, Kruskal-Wallis with Dunn’s Multiple Comparison test. All other comparisons were not significant.</p

Histopathology scores in mice.

a<p>Cellular infiltrate (0–6): 0, none; 1 mild, multifocal; 2 mild, widespread or moderate multifocal; 3, mild widespread and moderate multifocal or severe multifocal; 4, moderate widespread; 5, moderate widespread and severe multifocal; and 6, severe widespread. Mucus metaplasia and functional atrophy (0–3): 0, absent; 1 mild; 2 moderate; and 3 severe.</p><p>*p<0.05, **p<0.01, c.f. unvaccinated/challenged group; One-way ANOVA with Dunnett’s Multiple Comparison Test</p

Recombinant EHV-1 gG purified from a baculovirus expression system.

<p>Purified EHV-1 gG from Sf9 cells infected with recombinant baculovirus, separated on 10% SDS-PAGE and (A) stained with Coomassie Brilliant Blue-250, or (B) transferred to PVDF membrane and probed with anti-EHV-1 gG rat polyclonal serum and HRP-conjugated goat anti-rat IgG.</p

Vaccine formulations delivered to BALB/c mice.

a<p>gG, glycoprotein G;</p>b<p>CT, cholera toxin.</p