Heterologous in Epigenetics Compound Library research buy vivo neutralization of mHK6a virus of genotype 6a was more effective than mED43 neutralization. Although a 10-fold higher inoculum (105 IU/mouse) was injected, half of the H06-treated mice were completely protected. However, this higher dose

was needed because a 5-fold lower dose (2 × 104 IU/mouse) of this isolate was not sufficient to establish a productive infection in all nontreated mice (data not shown). Even though we showed here that polyclonal antibodies isolated from Patient H can prevent or at least delay a heterologous infection in vivo, the efficacy of neutralization was less than what could be expected based on previous in vitro infections of cell cultures.14 In fact, those in vitro studies indicated that H06-cross-genotype neutralization would be 10- to 100-fold more effective than homologous neutralization. The reason for this discrepancy is still under investigation; however, one could anticipate

that the differences in structural characteristics between in vitro and in vivo produced virus could play a role. To exclude the possibility that the lack of protection was caused by escape mutations, we sequenced the complete envelope region of the Pim inhibitor seven H06-treated mice that became infected with mED43 or mHK6a viruses and compared the amino acid sequence with those of viruses isolated from control animals and the original viral inocula. In four animals we did not observe any amino acid mutations in the envelope sequence using a direct sequencing 上海皓元医药股份有限公司 method. The E1-sequence was completely conserved in all but one H06-treated animals. In this mED43-infected mouse we detected an L221M mutation. Because this mutation

was also detected in one of the control animals it is unlikely to be the result of viral escape. In fact, this mutation corresponds to the wildtype sequence retrieved from the patient virus from which this challenge virus originated (Y11604).27 In one H06-treated animal a single mutation in the HVR1-region of the E2 protein was observed (S405P). It is doubtful that this mutation would provoke resistance to neutralization because antibodies that target HVR-1 usually are isolate-specific. Likewise, another mutation (N573T) was observed in the variable intergenotypic region of E2, again arguing for spontaneous mutation. We also observed a mutation at position 448 in one HK6a-infected mouse (N448D), which is a known glycosylation site within E2. This is surprising because it has been shown by Helle et al.28 that a loss of glycosylation renders the virus more sensitive to neutralizing antibodies. In general, none of the mutations we observed are located in previously reported conserved neutralizing epitopes. Using our direct sequencing approach it remains possible that we missed certain mutations that are only present in a minor fraction of the virus pool.