Peptide blockade of 11–88×8 antiserum.

<p>A. Sera of Balb/c mice immunized three times with 11–88×8 or 11–88×8Δ using Alum+MPL as adjuvant were harvested two weeks after the final boost. The antisera diluted 1∶100 were reacted with microtiter plates coated with 1 µg of two HPV16 L2 peptides encompassing residues 43–62 and 48–67. After washing, specific reactivity was measured by ELISA using peroxidase-linked anti-mouse IgG. B. Pooled sera of Balb/c mice immunized three times with 11–88×8 using Alum+MPL as adjuvant were harvested either pre-immunization (Pre-bleed) or two weeks after the final boost (×8 Sera). The antiserum to 11–88×8 was diluted 1∶50 in a total volume of 200 µl and incubated with 7 µg of HPV16 L2 peptide encompassing residues 13–32, 18–37, 23–42, 28–47, 33–52, 38–57, 43–62, 48–67, 53–72, 58–77, 63–82 or 71–90 for an hour prior to mixing with HPV16 pseudovirions carrying a SEAP reporter for a further hour at ambient temperature and subsequent infection of 293TT cells. Infection was measured as optical density, and only HPV16 L2 pepitdes 13–32 and 18–37 substantially blocked neutralization by 1∶3200 dilution of antiserum to 11–88×8. C. Sera of Balb/c mice immunized three times with 11–88×8 using Alum+MPL as adjuvant were harvested two weeks after the final boost and pooled (×8). The 11–88×8 antiserum was mixed with two HPV16 L2 peptides encompassing residues 43–62 and 48–67 (+Peptide) in the same ratios as in B. The 11–88×8 antiserum was administered i.p. either alone or pre-mixed with peptide in volumes of 20 µl, 5 µl or 2 µl to naïve mice (in groups of 5). Separate groups of mice received 200 µg each of antibody affinity purified with protein G columns from the sera of rabbits hyper-immunized with HPV16 L2 peptides 17–36, 47–66 or 373–392. All groups of mice (except the Background group) were subsequently challenged intra-vaginally with HPV16 pseudovirions carrying a luciferase reporter. Infection was assessed by measuring bioluminescence three days later.