Matrix-M™ adjuvation broadens protection induced by seasonal trivalent virosomal influenza vaccine

Background: Influenza virus infections are responsible for significant morbidity worldwide and therefore it remains a high priority to develop more broadly protective vaccines. Adjuvation of current seasonal influenza vaccines has the potential to achieve this goal. Methods: To assess the immune potentiating properties of Matrix-M (TM), mice were immunized with virosomal trivalent seasonal vaccine adjuvated with Matrix-M (TM). Serum samples were isolated to determine the hemagglutination inhibiting (HAI) antibody titers against vaccine homologous and heterologous strains. Furthermore, we assess whether adjuvation with Matrix-M (TM) broadens the protective efficacy of the virosomal trivalent seasonal vaccine against vaccine homologous and heterologous influenza viruses. Results: Matrix-M (TM) adjuvation enhanced HAI antibody titers and protection against vaccine homologous strains. Interestingly, Matrix-M (TM) adjuvation also resulted in HAI antibody titers against heterologous influenza B strains, but not against the tested influenza A strains. Even though the protection against heterologous influenza A was induced by the adjuvated vaccine, in the absence of HAI titers the protection was accompanied by severe clinical scores and body weight loss. In contrast, in the presence of heterologous HAI titers full protection against the heterologous influenza B strain without any disease symptoms was obtained. Conclusion: The results of this study emphasize the promising potential of a Matrix-M (TM)-adjuvated seasonal trivalent virosomal influenza vaccine. Adjuvation of trivalent virosomal vaccine does not only enhance homologous protection, but in addition induces protection against heterologous strains and thus provides overall more potent and broad protective immunit

Matrix-M™ adjuvant enhances immunogenicity of both protein- and modified vaccinia virus Ankara-based influenza vaccines in mice

Influenza viruses continuously circulate in the human population and escape recognition by virus neutralizing antibodies induced by prior infection or vaccination through accumulation of mutations in the surface proteins hemagglutinin (HA) and neuraminidase (NA). Various strategies to develop a vaccine that provides broad protection against different influenza A viruses are under investigation, including use of recombinant (r) viral vectors and adjuvants. The replication-deficient modified vaccinia virus Ankara (MVA) is a promising vaccine vector that efficiently induces B and T cell responses specific for the antigen of interest. It is assumed that live vaccine vectors do not require an adjuvant to be immunogenic as the vector already mediates recruitment and activation of immune cells. To address this topic, BALB/c mice were vaccinated with either protein- or rMVA-based HA influenza vaccines, formulated with or without the saponin-based Matrix-M™ adjuvant. Co-formulation with Matrix-M significantly increased HA vaccine immunogenicity, resulting in antigen-specific humoral and cellular immune responses comparable to those induced by unadjuvanted rMVA-HA. Of special interest, rMVA-HA immunogenicity was also enhanced by addition of Matrix-M, demonstrated by enhanced HA inhibition antibody titres and cellular immune responses. Matrix-M added to either protein- or rMVA-based HA vaccines mediated recruitment and activation of antigen-presenting cells and lymphocytes to the draining lymph node 24 and 48 h post-vaccination. Taken together, these results suggest that adjuvants can be used not only with protein-based vaccines but also in combination with rMVA to increase vaccine immunogenicity, which may be a step forward to generate new and more effective influenza vaccines

Immunogenicity of a recombinant hemagglutinin neuraminidase-Porcine rubulavirus produced by Escherichia coli of Porcine rubulavirus gives protective immunity of litter after challenge

Porcine rubulavirus (PRV) is a contagious virus that affects the Mexican swine industry. This work aimed to evaluate the immunogenicity of an recombinant hemagglutinin neuraminidase-Porcine rubulavirus (rHN-PorPV) candidate vaccine on pregnant sows, and the protective efficacy afforded to their 7-day- old suckling piglets against PRV lethal challenge. Three sows were immunized with rHN-PorPV formulated with immune-stimulating complex (ISCOMs) and two sows with rHN- PorPV protein alone as well as a mock-immunized pregnant sow (negative control). Quantitative ELISA detected a high concentration of anti-rHN-PorPV Immunoglobulin G (IgG) antibodies in sow sera after the second dose of vaccine administered on day 14 until farrowing, showing viral-neutralizing and cross-neutralization activity against different variants of PRV. Sera samples from piglets of immunized sows (with or without adjuvant), showed high concentrations of IgG antibodies. As expected, piglets from the negative control sow (n=5), exhibited severe signs of disease and 100% of mortality after PRV challenge study. Conversely, 75% and 87.5% of the piglets born from the rHN-PorPV and the rHN-PorPV-ISCOMs-immunized sows (n= 8), survived, respectively, showing milder PRV clinical signs. Our data indicate that rHN-PorPV candidate vaccine produced in Escherichia coli induces efficient humoral response in pregnant sows and that the maternally derived immunity provides high protection to suckling piglets against PRV lethal challenge

Matrix-M™ adjuvant induces local recruitment, activation and maturation of central immune cells in absence of antigen.

Saponin-based adjuvants are widely used to enhance humoral and cellular immune responses towards vaccine antigens, although it is not yet completely known how they mediate their stimulatory effects. The aim of this study was to elucidate the mechanism of action of adjuvant Matrix-M™ without antigen and Alum was used as reference adjuvant. Adjuvant Matrix-M™ is comprised of 40 nm nanoparticles composed of Quillaja saponins, cholesterol and phospholipid. BALB/c mice were subcutaneously injected once with, 3, 12 or 30 µg of Matrix-M™, resulting in recruitment of leukocytes to draining lymph nodes (dLNs) and spleen 48 h post treatment. Flow cytometry analysis identified CD11b(+) Gr-1(high) granulocytes as the cell population increasing most in dLNs and spleen. Additionally, dendritic cells, F4/80(int) cells, T-, B- and NK-cells were recruited to dLNs and in spleen the number of F4/80(int) cells, and to some extent, B cells and dendritic cells, increased. Elevated levels of early activation marker CD69 were detected on T-, B- and NK-cells, CD11b(+) Gr-1(high) cells, F4/80(int) cells and dendritic cells in dLNs. In spleen CD69 was mainly up-regulated on NK cells. B cells and dendritic cells in dLNs and spleen showed an increased expression of the co-stimulatory molecule CD86 and dendritic cells in dLNs expressed elevated levels of MHC class II. The high-dose (30 µg) of Matrix-M™ induced detectable serum levels of IL-6 and MIP-1β 4 h post administration, most likely representing spillover of locally produced cytokines. A lesser increase of IL-6 in serum after administration of 12 µg Matrix-M™ was also observed. In conclusion, early immunostimulatory properties were demonstrated by Matrix-M™ alone, as therapeutic doses resulted in a local transient immune response with recruitment and activation of central immune cells to dLNs. These effects may play a role in enhancing uptake and presentation of vaccine antigens to elicit a competent immune response

Innate immune responses induced by the saponin adjuvant Matrix-M in specific pathogen free pigs

Abstract Saponin-based adjuvants have been widely used to enhance humoral and cellular immune responses in many species, but their mode of action is not fully understood. A characterization of the porcine transcriptional response to Matrix-M was performed in vitro using lymphocytes, monocytes or monocyte-derived dendritic cells (MoDCs) and in vivo. The effect of Matrix-M was also evaluated in specific pathogen free (SPF) pigs exposed to conventionally reared pigs. The pro-inflammatory cytokine genes IL1B and CXCL8 were up-regulated in monocytes and lymphocytes after Matrix-M exposure. Matrix-M also induced IL12B, IL17A and IFNG in lymphocytes and IFN-α gene expression in MoDCs. Several genes were indicated as up-regulated by Matrix-M in blood 18 h after injection, of which the genes for IFN-α and TLR2 could be statistically confirmed. Respiratory disease developed in all SPF pigs mixed with conventional pigs within 1–3 days. Two out of four SPF pigs injected with saline prior to contact exposure displayed systemic symptoms that was not recorded for the four pigs administered Matrix-M. Granulocyte counts, serum amyloid A levels and transcription of IL18 and TLR2 coincided with disease progression in the pigs. These results support further evaluation of Matrix-M as a possible enhancer of innate immune responses during critical moments in pig management

Median fluorescence intensity (MFI) of CD86 and MHC class II on CD19⁺ or CD11c⁺ cells in spleen post Matrix-M™ treatment.

<p>Mice were injected subcutaneously at the base of the tail and the spleen was harvested and analyzed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041451#s2" target="_blank">Materials and Methods</a>. Data is shown as mean ± SD (n = 6–8 mice). Significant differences from PBS treatment using Kruskal-Wallis test with Dunn's posttest are outlined with *, <i>p<0.05</i>; **, <i>p<0.01</i>; ***, <i>p<0.001</i>.</p

Expression of CD69 after Alum treatment.

<p>1% Alum was injected subcutaneously at the base of the tail and at 24 or 48 h post treatment cells were prepared and the expression of CD69 on cells from the draining lymph nodes (dLNs) (A and B) or the spleen (C and D) were analyzed. Data is presented as mean ± SD (n = 3–6 mice). Significant differences between Alum treatment and naïve mice using Mann-Whitney test are outlined with *, <i>p<0.05</i>; **, <i>p<0.01</i> ; ***, <i>p<0.001</i>.</p

High-dose Matrix-M™ increases the IL-6 (A) and MIP-1β (B) levels in serum.

<p>Mice were subcutaneously injected at the base of the tail with 3, 12 or 30 µg Matrix-M™ or PBS. Cytokine levels in serum were detected by Cytometric Bead Array (CBA) 4 h after injection. Data is presented as mean ± SD (n = 6–8 mice). Significant differences using Kruskal-Wallis test with Dunn's posttest are outlined with *, <i>p<0.05</i>; **, <i>p<0.01</i>; ***, <i>p<0.001</i>. M, Matrix-M™.</p

Fold change in total cell numbers in dLNs and spleen after Matrix-M™ or Alum treatment.

<p>Alum or 3, 12 or 30 µg Matrix-M™ were injected subcutaneously at the base of the tail. After 48 h cells from the two draining lymph nodes (dLNs) (A) and the spleen (B) were prepared and total cell counts were performed. The relative difference in cell numbers between 3, 12 or 30 µg Matrix-M™- and PBS-treated mice or Alum-treated and naïve mice, respectively are presented. Dotted line represent no difference in cell number between treated and control mice (fold change = 1). Data is shown as mean ± SD (n = 6–8 mice). Significant differences between Matrix-M™ - and PBS-treated mice using Kruskal-Wallis test with Dunn's posttest or between Alum-treated and naïve mice using Mann-Whitney test are outlined with *, <i>p<0.05</i>; **, <i>p<0.01</i>; ***, <i>p<0.001</i>. M, Matrix-M™.</p