In the experiments described here, we found a lack of correlation between the inhibitory effects of virus-specific antibody in vitro and in vivo. We found that although both polyclonal antibodies and MAbs failed to neutralize the AdCDQ vector in a standard in vitro assay, they nonetheless prevented effective immunization with that vector in vivo.
The detrimental effect of AdHu5-specific VNAs on transgene product-specific B-and T-cell responses has been well documented in experimental animal models (2
). Consistent with this, in a recent clinical trial of an AdHu5-based HIV-1 vaccine (the STEP Trial), levels of CD8+
T-cell responses to HIV-1 antigens (as measured by IFN-γ enzyme-linked immunospot assays) were reduced in subjects with high levels of AdHu5-specific VNAs (9a
). Furthermore, although there did not appear to be a specific correlation between infection and a low-level CD8+
T-cell response, there was a possible trend toward increased infection in vaccinees with higher levels of preexisting VNAs (1a
). The results of the STEP trial are not yet understood, and it remains to be determined whether vaccine-induced CD8+
T cells can ameliorate HIV infection in humans. Nonetheless, it is clear that Ad-specific neutralizing antibodies present a barrier to immunization with Ad vectors.
To circumvent the problem of preexisting VNAs, a number of investigators have developed chimeric AdHu5 vectors in which critical portions of the hexon are replaced by hexon sequences from other Ad serotypes and have found that such chimeras escape neutralization by AdHu5-induced VNAs (12
). An AdHu5 chimera in which exposed hexon loops were replaced by those of AdHu48 was recently demonstrated to escape neutralization by AdHu5-specific antibody and to provide effective immunization in the face of preexisting immunity to AdHu5 (12
). However, our results suggest that in vitro assays may not reliably predict the effects of antiviral antibodies in vivo.
Viral neutralization escape mutants selected by growth in monoclonal neutralizing antibodies often have single-site mutations that significantly reduce (but do not necessarily eliminate) the binding affinity of individual antibodies. To create the AdCDQ vector, we replaced 3 amino acids within a hexon surface loop recognized by a panel of MAbs including MAb 4C1 (10
). We found that 4C1, despite its inability to neutralize AdCDQ in vivo, bound to AdCDQ in an ELISA (although its avidity appeared to be significantly reduced). Interestingly, MAb 4D1, which recognizes a different epitope within hexon, bound efficiently in the ELISA but did not neutralize virus in vitro or inhibit virus in vivo.
The mechanism by which Ads are neutralized is not certain: antibodies may aggregate virions, block interactions with cell surface receptors, or interfere with postentry events in virus replication (20
). Even less is known about antibody-mediated effects in vivo. We found that a MAb that does not neutralize AdCDQ in vitro nonetheless prevented transgene delivery and the induction of transgene-specific CD8+
T-cell responses in vivo. Neutralization of AdCDQ in vitro was not potentiated by murine serum or plasma, nor was inhibition in vivo attenuated by the elimination of Fc receptor function.
We do not know the mechanism by which anti-AdC68 antibody inhibits effective immunization with the AdCDQ vector, but it is clear that antibody prevented the transduction of both muscle cells and dendritic cells at the site of immunization. The in vivo environment is much more complex than that encountered in a neutralization assay. Our data suggest that in vitro neutralization assays, despite their convenience and widespread use, cannot replace in vivo trials for the assessment of new viral vectors.