Chimeric Rhinoviruses Displaying MPER Epitopes Elicit Anti-HIV Neutralizing Responses

Background: The development of an effective AIDS vaccine has been a formidable task, but remains a critical necessity. The well conserved membrane-proximal external region (MPER) of the HIV-1 gp41 glycoprotein is one of the crucial targets for AIDS vaccine development, as it has the necessary attribute of being able to elicit antibodies capable of neutralizing diverse isolates of HIV. Methodology/Principle Findings: Guided by X-ray crystallography, molecular modeling, combinatorial chemistry, and powerful selection techniques, we designed and produced six combinatorial libraries of chimeric human rhinoviruses (HRV) displaying the MPER epitopes corresponding to mAbs 2F5, 4E10, and/or Z13e1, connected to an immunogenic surface loop of HRV via linkers of varying lengths and sequences. Not all libraries led to viable chimeric viruses with the desired sequences, but the combinatorial approach allowed us to examine large numbers of MPERdisplaying chimeras. Among the chimeras were five that elicited antibodies capable of significantly neutralizing HIV-1 pseudoviruses from at least three subtypes, in one case leading to neutralization of 10 pseudoviruses from all six subtypes tested. Conclusions: Optimization of these chimeras or closely related chimeras could conceivably lead to useful components of an effective AIDS vaccine. While the MPER of HIV may not be immunodominant in natural infection by HIV-1, its presence in a vaccine cocktail could provide critical breadth of protection

INVOLVEMENT OF THE TRANSCRIPTION TERMINATION FACTOR, RHO, IN THE REGULATION OF DNA SUPERCOILING IN ESCHERICHIA COLI: AN EXTENDED MODEL FOR RHO-MEDIATED TRANSCRIPTION TERMINATION

Much remains unknown about how the Rho protein causes specific termination of transcription and release of the transcription complex and RNA in bacteria. I was intrigued by the suggestion that Rho might carry out these functions by the affecting the topology of DNA. The studies described indicate that most rho mutants examined had reduced supercoiling of plasmid DNA relative to rho \sp+ counterparts. Transductional studies with the strongly defective rho-15 mutant indicated that this phenomenon is a function of the rho allele (and not of a secondary mutation). Supercoiling of nucleoids was examined; rho-15 nucleoids apparently had relatively less folded genomic DNA than rho \sp+ nucleoids. The cell\u27s topoisomerases, DNA gyrase and DNA topoisomerase I, were found to be expressed at normal levels in the rho-15 mutant. Levels of three other proteins examined all showed altered levels in rho-15 extracts. The altered levels of two of these, the SSB protein and DNA polymerase I, or of unexamined proteins could be responsible for the reduced plasmid supercoiling in rho \sp- cells. Alternatively, the remarkable similarity seen between rho and gyrB mutants suggests a central relationship between these proteins. Furthermore, normalization of transcription in rho\sp- strains with suppressor mutations was matched in every case by normalization of DNA supercoiling. This suggested a mechanistic connection between transcription termination and DNA supercoiling. To account for this, I propose the following model. When Rho protein is at the termination site, it can either directly or by stimulating RNA polymerase cause an unwinding of the DNA. The result would be to block further transcription and favor dissociation of the transcription complex and RNA from the DNA. Some of the time, DNA gyrase would be expected to respond to the transient unwinding (and resultant loss of local negative superhelicity) by introducing new superhelical turns. If mutant Rho could not induce Rho-mediated unwinding, there would be no need for a response by gyrase, possibly explaining why plasmid supercoiling in most rho mutants is reduced relative to wild type cells. The implications of this model are that the consequences of mutation in rho are derived from both transcription termination defects and reduced supercoiling