3B). The median intra-species group

surface-exposed loop genetic distances for these Alpha-7 and Alpha-9 L1 sequences were similar at 0.19 (IQR 0.15–0.20) and 0.24 (0.18–0.24), respectively (p = 0.146), and substantially lower than the median inter-species genetic distance of 0.37 (0.35–0.40; p < 0.001). Within the Alpha-9 species group, the antigenic similarity between HPV33 and HPV58 is perhaps reflected in the low genetic distance between these genotypes. The apparent antigenic relationship between HPV39 and HPV59 within the Alpha-7 species group, however, is not similarly reflected by low genetic distances. There C59 wnt nmr were other sporadic instances of weaker cross-neutralization, for example between HPV16, HPV31 and HPV33. Interpretation of these weaker responses, however, has to be tempered by the observation that three of the

thirty-six rabbits generated weak inter-species responses: two animals immunized with HPV31 VLP (one with cross-reactivity against HPV18 and one against HPV68) and one animal immunized with HPV35 VLP (cross-reactivity against HPV45, HPV59 and HPV68). Weak intra-species group responses are intuitively likely to be genuine, but given the inter-species genetic distances in the surface-exposed loops (Fig. 3B) weak inter-species responses should be interpreted GW3965 in vivo with some caution. Pre-immune sera were negative for neutralizing antibodies against all Alpha-7 and Alpha-9 HPV pseudoviruses and the control BPV (data not shown). A tetravalent preparation containing HPV16, HPV18, HPV39 and HPV58 VLP was used to immunize a group of five NZW rabbits following the same schedule as that for the individual immunizations (Fig. 4). All five over rabbits generated high titer neutralizing antibodies against the immunizing genotypes HPV16, HPV18, HPV39 and HPV58 and the titers were similar to those obtained when used as individual immunogens with median individual

and tetravalent type-specific neutralization titers for HPV16 (80,813 vs. 161,025), HPV18 (21,941 vs. 17,637), HPV39 (86,678 vs. 53,612) and HPV58 (140,129 vs. 105,258) as indicated (Fig. 2 and Fig. 4). Conversely, the breadth of cross-neutralization seen against the Alpha-7 and particularly the Alpha-9 pseudoviruses was greater than when VLP were used individually: all five rabbits generated neutralizing antibodies against HPV33 and three to four of five rabbits also generated neutralizing antibodies against HPV31, HPV35, HPV45, HPV52, HPV59 and HPV68. None of the five rabbits generated antibodies capable of neutralizing BPV and pre-immune sera were negative for neutralizing antibodies against all Alpha-7 and Alpha-9 HPV pseudoviruses. To establish which of the HPV16 and/or HPV58 VLP immunogen(s) were responsible for the generation of the cross-neutralizing antibody responses against HPV31 and HPV33 we used VLP as competing antigens in neutralization tests (Table 1 and Supplemental Fig.