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Archived Comments for: Antigenic presentation of heterologous epitopes engineered into the outer surface-exposed helix 4 loop region of human papillomavirus L1 capsomeres

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  1. Differences and similarities between our work and those of Varsani, et al. (J. Virol 2003: 77; 8386-8393)

    Yoshihiko Murata, University of Rochester School of Medicine and Dentistry

    8 September 2009

    We do note that Varsani, et al. (J. Virol 2003: 77; 8386-8393) have previously engineered a single 13 aa neutralizing epitope from HPV 16 L2 protein within various regions of the HPV 16L1 protein. One of their L1 derivatives (ChiΔF-L2) bore the L2 epitope as a substitution of aa 414-426, which maps to the L1 helix 4 (h4) domain. As we have found with our capsomere derivatives, ChiΔF-L2 reacts slightly more strongly with H16:V5 mAb in ELISAs as compared to intact 16L1 VLPs. When injected into laboratory mice, ChiΔF-L2 was the most immunogenic among the various L1-L2 chimeras with respect to the L2 epitope and as assayed by immunoblots to detect reactogenicity against bacterially derived L2 protein; our findings confirm their assertion that h4 region is “probably highly immunogenic” (p. 8392). There are no data reported on the HPV pseudoneutralization capacities of the resulting sera.

    However, our constructions differ from that of Varsani, et al. in that: 1) using baculovirus-derived L1 derivatives, we confirm previous results from bacterially derived L1 deletions that the removal of a larger portion of L1 (aa 404-436) results in capsomere formation; and 2) we demonstrate that the replacement of such a deletion with one of two neutralizing epitopes of varying length and predicted in-solution structure (aa 255-278, which forms a helix-coil-helix, and aa 423-436, a linear epitope) of the respiratory syncytial virus (RSV) fusion (F) protein also leads to capsomeres with some degree of aggregation as confirmed by sucrose gradient ultracentrifugation. Thus, we show that the larger h4 deletion that that used to generate ChiΔF-L2 can accommodate the presence of heterologous aa sequences with minimal to moderate ultrastructural perturbations of L1 capsomeres.

    In generating our constructions, we removed the L1 Cys 428, which is involved in intercapsomeric disulfide bond formation and is retained in ChiΔF-L2. We also note that as compared to electron micrographs of our capsomere preparations, those of ChiΔF-L2 qualitatively appear to exhibit increased degree of aggregation (Figure 2, p. 8389); no assays of in-solution behavior of ChiΔF-L2 are presented. Such aggregation may be related to residual and potentially aberrant disulfide formation among L1 oligomers via Cys428 within each L1 monomer. Thus, in our current paper, we confirm and extend previous finding by Varsani, et al.

    Competing interests

    YM, RCR, and EEW are authors of a provisional patent
    application on the use of human papillomavirus L1 protein and its derivatives, including capsomeres, as RSV vaccine candidates. This is clearly stated in our paper and is reproduced here.

  2. Is this original?

    Edward Rybicki, University of Cape Town

    8 September 2009

    Dear Dr Murata;

    we were very interested to read your recent paper in Virology Journal describing h4 insertions / replacements in HPV-16 L1 protein as a means of displaying heterologous peptides - especially as you chose this region because lack of it interferes with capsid formation and presents peptides well.

    We were a little surprised, though, to see this in the abstract:

    "HPV L1 monomers bearing heterologous epitopes within the L1 h4 region can self-assemble into capsomeres that elicit antibody response against such non-
    HPV encoded epitopes. Thus, the L1 h4 region can function as a novel antigen display site within the L1 pentamer, which in turn may serve as a potential
    vaccine template. "

    and this in the Discussion:

    "Because papillomavirus L1 protein-based oligomers (capsomeres and VLPs) can elicit a broad array of immune responses, they have been studied as potential
    vaccine platforms (reviewed in [15]). Such efforts have primarily focused on placing heterologous epitopes on surface-displayed, genetically variable loops of L1 or at the carboxy-terminus of full-length or truncated L1. Since capsomeres are also immunogenic, we tested the hypothesis that the h4 domain of the L1 monomer, which projects laterally and outwards on L1 pentamers, can be used for antigenic display of foreign epitopes. We demonstrate that: 1) such antigen presentation is feasible and does not overtly affect the formation of capsomeres; 2) such capsomeres likely exists as monomeric capsomeres with some degree of aggregation noted; and 3) mice immunized with capsomeres bearing RSV F epitopes generate antisera that recognizes the purified F protein. Thus, we conclude that foreign epitopes embedded within the h4 domain can be immunogenic when presented in the context of capsomeres."

    ...given that a paper from our group in 2003 [Varsani et al., J Virol 77: 8386-8393, 2003] described much the same thing. We said then:

    "The most immunogenic chimera overall was ChiF-L2, where the L2 epitope [L2 108-120] was presented in place of the h4 helix: this region (aa 414 to 426) is highly exposed in the atomic structure of HPV-16 L1 proposed by Modis et al. (24) and therefore probably highly immunogenic. Chimera ChiF-L2 bound all the neutralizing MAbs and induced a strong L1 and L2 epitope response. We suspect that the h4 helix in native VLPs forms important interactions and that altering this region prevents the formation of VLPs. Chen et al. (4) showed that deletion of the region aa 408 to 431 resulted in pentamer formation only; these observations support our finding that ChiF-L2 is only seen to form capsomers." [bold = my emphases]

    Do you have any comment on this? As it is, your paper appears to reinvent a wheel already well described some 6 years ago!

    Competing interests

    We have published a paper (Varsani et al., J Virol 77: 8386-8393, 2003) which reached many of the same conclusions described here - and have patented L1 peptide display using the h4 loop (WO/2003/097673).