In Situ Multiplexing to Identify, Quantify, and Phenotype the HIV-1/SIV Reservoir Within Lymphoid Tissue.

Modern combination antiretroviral therapy (ART) regimens provide abiding viral suppression for most individuals infected with human immunodeficiency virus (HIV). However, the persistence of viral reservoirs ensures that eradication of HIV-1 (i.e., cure) or sustained ART-free remission (i.e., functional cure) remains elusive, necessitating continual, strict ART adherence and contributing to HIV-1-related comorbidities. Eradication of these viral reservoirs, which persist primarily within lymphoid tissue, will require a deeper understanding of the cellular neighborhoods in which latent and active HIV-1-infected cells reside. By pairing highly sensitive in situ hybridization (ISH) with an exceptionally flexible immunofluorescence (IF) approach, we describe a simple, yet highly adaptable multiplex protocol for investigating the quantity, distribution, and characteristics of HIV-1 viral reservoirs.

Development of an Acid-Resistant Salmonella Typhi Ty21a Attenuated Vector For Improved Oral Vaccine Delivery.

The licensed oral, live-attenuated bacterial vaccine for typhoid fever, Salmonella enterica serovar Typhi strain Ty21a, has also been utilized as a vaccine delivery platform for expression of diverse foreign antigens that stimulate protection against shigellosis, anthrax, plague, or human papilloma virus. However, Ty21a is acid-labile and, for effective oral immunization, stomach acidity has to be either neutralized with buffer or by-passed with Ty21a in an enteric-coated capsule (ECC). Several studies have shown that efficacy is reduced when Ty21a is administered in an ECC versus as a buffered liquid formulation, the former limiting exposure to GI tract lymphoid tissues. However, the ECC was selected as a more practical delivery format for both packaging/shipping and vaccine administration ease. We have sought to increase Ty21a acid-resistance to allow for removal from the ECC and immune enhancement. To improve Ty21a acid-resistance, glutamate-dependent acid resistance genes (GAD; responsible for Shigella spp. survival at very low pH) were cloned on a multi-copy plasmid (pGad) under a controllable arabinose-inducible promoter. pGad enhanced acid survival of Ty21a by 5 logs after 3 hours at pH 2.5, when cells were pre-grown in arabinose and under conditions that promote an acid-tolerance response (ATR). For genetically 100% stable expression, we inserted the gad genes into the Ty21a chromosome, using a method that allowed for subsequent removal of a selectable antibiotic resistance marker. Further, both bacterial growth curves and survival assays in cultured human monocytes/macrophages suggest that neither the genetic methods employed nor the resulting acid-resistance conferred by expression of the Gad proteins in Ty21a had any effect on the existing attenuation of this vaccine strain.