Induction of IgG titers in CRs immunized with VLPs and the virus.

<p>RSV-specific IgG levels in serum samples of CRs immunized twice were measured as already described. The histogram shows the results (mean ± SD, n = 3) of samples collected on days 0, 21, 42 and 46 for each of the started groups: IgG levels of adjuvanted VLPs on days 42 and 46 were significantly higher by both IM and SC routes compared to those induced by the virus and VLPs.</p

CRs immunized with adjuvanted RSV VLPs protected the lower as well as the upper respiratory tract.

<p><b>A:</b> Vaccination with RSV VLPs provided virtually no protection in the lung or nose of CRs: There was a statistically significant, but not a meaningful reduction in the virus load in the lung (<b>Gp1</b> vs <b>Gp3</b>, <i>P</i><<i>0</i>.<i>019</i>; <b>Gp1</b> vs <b>Gp4</b>, <i>P<0</i>.<i>029)</i> and no virus reduction in nasal wash samples (<i>P>0</i>.<i>05)</i>. <b>B:</b> Adjuvanted RSV VLPs conferred protection based on substantial virus clearance from the lung (<b>Gp1</b> vs <b>Gp3</b> and <b>Gp4</b>, <i>P<0</i>.<i>00001</i>) as well the nose of these animals (<b>Gp1</b> vs <b>Gp3</b><i>P<0</i>.<i>0073</i>; <b>Gp1</b> vs <b>Gp4</b>. <i>P<0</i>.<i>0013)</i>.</p

Conservation of RSV F protein antigenic sites characterized to date.

<p>Four antigenic group A and three group B RSV F protein sequences were aligned using ClustalW and visualized with Geneious Pro software. The RSV A2 F (Genbank protein accession ACO83301) serves as the reference strain. The other representative strains are A/1998/12-21 (Nashville, Tennessee, US, accession AFM95385), A/RSV-12 (Denver, Colorado, US, 2004–2005, accession AEO45929), A/Riyadh/2009 (Riyadh, Saudi Arabia, accession AEO23052), B/9320 (Massachusetts, US, 1977, accession AAR14266), B/NH1276 (New Haven, Connecticut, US, 2002, accession AFD34264), and B/TX11-56 (Dallas, Texas, US, 2011, accession AFD34265). Positions of antigenic sites 0, I, II, and IV are from reference <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004016#ppat.1004016-McLellan1" target="_blank">[15]</a>.</p

Induction of IgG titers in CRs immunized with VLPs and the virus.

<p>RSV-specific IgG levels in serum samples of CRs immunized twice were measured as already described. The histogram shows the results (mean ± SD, n = 3) of samples collected on days 0, 21, 42 and 46 for each of the started groups: IgG levels of adjuvanted VLPs on days 42 and 46 were significantly higher by both IM and SC routes compared to those induced by the virus and VLPs.</p

Co-expression of G, F and M proteins resulted in large quantities of RSV VLPs which were functionally assembled and immunoreactive: VLPs were purified as described, viewed by EM to evaluate their morphology and stained by immunogold-labelling technique to test for immune reactivity.

<p>(a) Arrow points to VLP-containing band in the sucrose gradient. (b) Several negatively stained particles resembling the parental virus; surface glycoproteins are easily seen. They resemble those reported for parental RSV by TEM (John Barr Lab., Leeds University, UK). (c) Immunoreactive gold-labelled particles stained with polyclonal RSV antibody and (d) those stained with RSV-F specific primary antibody are clearly seen. (e) Is a negative control where the primary antibody was omitted and VLPs were stained only with the gold-labelled secondary antibody.</p

CRs immunized with adjuvanted RSV VLPs induce strong NtAb response to RSV/A2, and RSV/B virus.

<p><b>A</b> and <b>C:</b> Show NtAb response (mean ± SD) against RSV-T/A2 and RSV/B respectively on stated days. The same figures show that VLPs alone induced NtAb response in only some of the animals and only weakly. <b>B</b> and <b>D:</b> Show neutralizing antibody response (mean ± SD) on day 42 against RSV-T/A2 and RSV/B respectively. Statistical difference between Gp 1 vs all other groups individually is also tabulated.</p

VLP-incorporated RSV proteins.

<p>The VLPs were harvested and purified as described and then processed and analyzed by SDS-PAGE. Panel A: The VLP-incorporated RSV proteins shown in two different representative preparations of purified VLPs were consistent in size to RSV M, F1 and G proteins. The precursor F0 protein and the F2 fragment were difficult to discern. An additional F protein band, a trimer, was visible in the VLPs and the virus in Panel C. The specificity of the RSV proteins in the VLPs was confirmed by the presence of equivalent sized proteins (kD) in RSV A2 virus (Panel C) and by their absence in the similarly processed “mock” particles (Panel B).</p

Vaccination schedule and experimental design.

<p>Thirty CRs (5 CRs/group; 6 groups), ~75–150 gm body weight were used. Body weight and sex distribution was similar across all groups. For subcutaneous (SC) vaccinations, 200 μL of vaccine was injected (using a tuberculin syringe) into subcutaneous space of the neck area. For intramuscular (IM) vaccinations, 100 μL of vaccine or diluent was injected (using a tuberculin syringe) into the area of each tibialis anterior (TA) muscle. Challenge virus was administered intranasally (100 μL) after CRs were lightly anesthetized with isoflurane.</p

RSV A2 and RSV VLP-induced cytokines IL-4, IL-5, IL-13, IL-10, IL12p70, IFN-ү, TNF-α and IL-6, and eotaxin, and the role of TL4 in their induction: PMA stimulated THP-1 cells were treated with RSV or RSV VLPs in presence or absence of anti-TLR-4 antibody.

<p>Both the virus and the VLPs upregulated Th1 cytokine TNF-α and proinflammatory cytokine IL6 while Th2 cytokines IL4, IL5 and IL-13 as well as eotaxin were not elevated. IL-10 was in the lower range. TLR-4 blockade reduced the production of TNF-α, IL-6 and IL-10. 2-way ANOVA: ***<i>p</i><0.0001</p

Current challenges and development of polyvalent vaccines.

<p><b>(A)</b> Current potential challenges to development of polyvalent vaccine preparations. (<b>B</b>) Development and licensing of polyvalent vaccines in the United States. Combination vaccines are not reviewed here. Different colors indicate vaccines against the same pathogen. Blue, <i>Streptococcus pneumoniae</i> (polysaccharide vaccine and conjugate vaccine); red, poliovirus (inactivated vaccine and oral, live-attenuated vaccine); purple, <i>Neissaeria meningitides</i> (meningococcal vaccine: polysaccharide and conjugate vaccine); yellow, rotavirus (live attenuated); green, HPV, VLP; gray, adenovirus; white, influenza (inactivated and live-attenuated vaccines). Not all influenza vaccines are included. HPV, human papillomavirus; QC, quality control; VLP, viruslike particle.</p