asa med Foreign polypeptide antigens can be incorporated

Foreign polypeptide antigens can be incorporated into VLPs to create “chimeric” structures, either as fusions to either end of the VLP capsid (coat) protein (CP) or as additions to well-presented loops [18]. This kind of direct fusion to the coat is usually good for small peptides, however, larger proteins may not be properly folded. In such cases, chemical conjugation to the VLP surface, usually through reactive amino or thiol groups, can be a better option [19], [20] as the folding of the coupled antigen is not dependent on how the VLP monomer folds and the site of attachment can be suitably engineered so that it does not cover important epitopes. The platform used here, derived from bacteriophage Qβ, is a 133 amino asa med CP (14.3 kDa), 180 copies of which self-assemble into icosahedral VLPs when expressed in E. coli. There are four externally-exposed amino groups on each subunit, which are available for conjugation to foreign antigens. Engineered versions of the Qβ CP have been used as vaccine candidates, with variable efficacy but consistently high safety [21], [22].
Materials and methods Brief descriptions are below. More information is provided in supplementary materials.
Results
Discussion In many cases, protective immunity against various viral and bacterial diseases has been achieved using live attenuated vaccines, because they closely mimic natural infection. However, for certain infections such as HIV, live attenuated viruses are not considered safe, both because of viral integration into the chromosome and because of mutations that can reactivate the virus. In such cases, virus-like particles or VLPs are an attractive alternative, as they resemble intact virions in terms of size and presentation of antigens, but are non-pathogenic, non-replicative and hence are safe for administration. Chimeric VLPs are an efficient way to display foreign epitopes in multivalent form and are able to elicit a robust immune response without adjuvant [37]. In this study, we displayed various designed gp120 fragment proteins on the surface of Qβ virus-like particles. We improved the stability and b12 affinity of our previously described fragment immunogen b122a [13] by incorporating the K383F mutation with additional disulfides. Construct b122a1-b resulted in the largest improvement in affinity to b12. No significant improvement was observed in the secondary structure of the protein as assessed by CD spectroscopy, but increased in vivo stability was seen in a β-lactamase activity assay [23]. For b122a-19iC conjugated particles, the final gp120 ELISA titers were the same both in absence and presence of adjuvant but increased titers against priming immunogen and neutralization was seen with the latter. This indicated that the adjuvant increased the immunogenicity of the particles. Sera from group 4 primed with the stabilized protein b122a1-b showed higher neutralization activity against Tier 1 viruses and also showed weak neutralization of the Tier 2, subtype B JRFL strain. Sera from the group primed with empty particles, showed low gp120 and priming immunogen titers, indicating that priming with the fragment immunogens is beneficial. Competition assays using both SPR and ELISA showed an increased CD4 binding site directed response in week 22 sera from group primed with Qβ VLPs displaying b122a1-b compared to other groups (Table 4, Fig. 2B). Collectively the data demonstrate that priming with a CD4bs directed fragment immunogen results in some degree of immune focusing to the CD4bs and improving immunogen stability and b12 binding affinity resulted in enhanced elicitation of neutralizing antibodies. However, a more native-like boosting immunogen than gp120 is likely required to elicit a higher proportion of neutralizing antibodies. Various studies attempting to focus the immune response to a particular epitope on HIV-1 Env have been carried out. Most of the immunogens described in these studies are based on the outer domain of gp120. An OD construct (OD1) containing residues, 252–482 from YU2 strain of gp120 was expressed in Drosophila S2 cells. This construct was glycosylated and also retained V1V2 and V3 variable loops, but the sera obtained after rabbit immunizations did not neutralize homologous YU2 virus [38]. In another study, mice were immunized with clade C OD as a fusion to human IgG1 Fc domain, but failed to elicit any neutralizing response. [39], [40]. Another study described two OD immunogens (monomeric and trimeric) based on group M consensus sequence. The immunogens were able to induce heterologous Tier 1 neutralizing responses although sera immunized with monomeric gp120 showed higher neutralization titers for all viruses [41]. The OD3 construct based on subtype C strain 1084i having stabilizing disulfides and a cavity filling mutation was able to elicit a Tier 1 and a weak Tier 2 neutralizing response in guinea pig immunization studies [42]. In the present work we were able to show elicitation of a strong cross-clade Tier 1 response with Qβ VLPs displaying either ODEC or, the stabilized b122a1-b even in the absence of adjuvant. b122a conjugated to Qβ VLP’s elicited a similar response when immunized in the presence of adjuvant. Sera elicited by Qβ VLP’s displaying the stabilized construct, b122a1-b, also elicited a weak Tier 2 response. One possible reason we did not see a better elicitation of antibodies that could neutralize multiple Tier 2 viruses in case of b122a VLP’s is that the displayed antigens might not have been conformationally intact. Another reason could be the fact that the valency of antigens displayed on the virus-like particles (Table S2) was lower than expected, resulting in reduced immunogenicity. Boosting with more native like trimeric gp140 [43] or trimeric gp120 [44], [45] instead of monomeric gp120 could prove beneficial, and can be used in future immunization studies to improve elicitation of neutralizing antibodies. VLP’s are known to enhance cell mediated responses. In this study we focused only on humoral responses and hence cannot comment on elicitation of T cell responses, which have been shown to substantially contribute to control of HIV-1 infection.