Flow cytometry assay modifications: Recommendations for method validation based on CLSI H62 guidelines.

The Clinical and Laboratory Standards Institute (CLSI) H62-Validation of Assays Performed by Flow Cytometry guideline, released in 2021, provides recommendations for platform workflow and quality system essentials, instrument setup and standardization, assay development and optimization and fit-for-purpose analytical method validation. In addition, CLSI H62 includes some recommendations for the validation strategies after a validated flow cytometric method has been modified. This manuscript builds on those recommendations and discusses the impact of different types of assay modifications on assay performance. Recommendations regarding which validation parameters to evaluate depending on the type of modification are provided. The impact of assay modification on the assay's intended use is discussed. When recommending minor deviations from the CLSI H62 process for a laboratory-initiated assay revision (e.g., specimen numbers for sensitivity, specificity, or precision studies), a rationale based on expert opinion is provided with the understanding that not every laboratory, assay type, and circumstance can be comprehensively addressed in this paper. These recommendations are meant as a practical recommendation and are not intended to be restrictive, prescriptive, or understood as necessarily sufficient to meet every specific requirement from regulatory bodies (e.g., FDA or New York State Department of Health).

A novel alphavirus vaccine encoding prostate-specific membrane antigen elicits potent cellular and humoral immune responses.

PURPOSE: Prostate-specific membrane antigen (PSMA) is an attractive target for active immunotherapy. Alphavirus vaccines have shown promise in eliciting immunity to tumor antigens. This study investigated the immunogenicity of alphavirus vaccine replicon particles (VRP) that encode PSMA (PSMA-VRP). EXPERIMENTAL DESIGN: Cells were infected with PSMA-VRP and evaluated for PSMA expression and folate hydrolase activity. Mice were immunized s.c. with PSMA-VRP or purified PSMA protein. Sera, splenocytes, and purified T cells were evaluated for the magnitude, durability, and epitope specificity of the anti-PSMA response. Antibodies were measured by flow cytometry, and cellular responses were measured by IFN-gamma enzyme-linked immunospot and chromium release assays. Cellular responses in BALB/c and C57BL/6 mice were mapped using overlapping 15-mer PSMA peptides. A Good Laboratory Practice-compliant toxicology study was conducted in rabbits. RESULTS: PSMA-VRP directed high-level expression of active PSMA. Robust T-cell and B-cell responses were elicited by a single injection of 2 x 10(5) infectious units, and responses were boosted following repeat immunizations. Anti-PSMA responses were detected following three immunizations with 10(2) infectious units and increased with increasing dose. PSMA-VRP was more immunogenic than adjuvanted PSMA protein. Responses to PSMA-VRP were characterized by Th-1 cytokines, potent CTL activity, and IgG2a/IgG2b antibodies. T-cell responses in BALB/c and C57BL/6 mice were directed toward different PSMA peptides. Immunogenic doses of PSMA-VRP were well tolerated in mice and rabbits. CONCLUSIONS: PSMA-VRP elicited potent cellular and humoral immunity in mice, and specific anti-PSMA responses were boosted on repeat dosing. PSMA-VRP represents a promising approach for immunotherapy of prostate cancer.

Enzymatic removal of mannose moieties can increase the immune response to HIV-1 gp120 in vivo.

The Env glycoproteins gp120 and gp41 are used in humoral immunity-based vaccines against human immunodeficiency virus (HIV-1) infection. One among many obstacles to such a vaccine is the structural defenses of Env glycoproteins that limit their immunogenicity. For example, gp120 mannose residues can induce immunosuppressive responses in vitro, including IL-10 expression, via mannose C-type lectin receptors on antigen-presenting cells. Here, we have investigated whether mannose removal alters gp120 immunogenicity in mice. Administering demannosylated gp120 (D-gp120) in the T(H)2-skewing adjuvant Alum induced approximately 50-fold higher titers of anti-gp120 IgG, compared to unmodified gp120. While the IgG subclass profile was predominantly T(H)2-associated IgG1, Abs of the T(H)1-associated IgG2a and IgG3 subclasses were also detectable in D-gp120 recipients. Immunizing with D-gp120 also improved T-cell responses. Giving an IL-10 receptor blocking MAb together with unmodified gp120 in Alum increased the anti-gp120 IgG titer, implicating IL-10 as a possible mediator of auto-suppressive responses to gp120.

Structural and immunogenicity studies of a cleaved, stabilized envelope trimer derived from subtype A HIV-1.

SOSIP gp140 trimers represent a soluble, stabilized, proteolytically cleaved form of the HIV-1 envelope (Env) glycoproteins. SOSIP gp140 derived from a subtype A HIV-1 isolate, KNH1144, forms exceptionally stable trimers that resemble virion-associated Env in antigenicity and topology. Here, we used electron microscopy to demonstrate that KNH1144 SOSIP gp140 trimers bound three soluble CD4 molecules in a symmetrical orientation similar to that seen for native Env. We compared the immunogenicities of KNH1144 SOSIP gp140 trimers and gp120 monomers in rabbits and found that the trimers were superior at eliciting neutralizing antibodies (NAbs) to homologous virus as well as neutralization-sensitive subtype B and C viruses. The NAb specificities for SOSIP antisera mapped in part to the CD4 binding site on gp120. We also observed adjuvant-dependent induction of antibodies to the residual levels of host cell proteins (HCPs) contained in the purified Env preparations. When present, HCP antibodies enhanced pseudovirus infection. Our findings are relevant for the further development of Env-based vaccines for HIV-1.

L-SIGN (CD209L) isoforms differently mediate trans-infection of hepatoma cells by hepatitis C virus pseudoparticles.

L-SIGN is a C-type lectin that is expressed on liver sinusoidal endothelial cells. Capture of Hepatitis C virus (HCV) by this receptor results in trans-infection of hepatoma cells. L-SIGN alleles have been identified that encode between three and nine tandem repeats of a 23 residue stretch in the juxtamembrane oligomerization domain. Here, it was shown that these repeat-region isoforms are expressed at the surface of mammalian cells and variably bind HCV envelope glycoprotein E2 and HCV pseudoparticles. Differences in binding were reflected in trans-infection efficiency, which was highest for isoform 7 and lowest for isoform 3. These findings provide a molecular mechanism whereby L-SIGN polymorphism could influence the establishment and progression of HCV infection.

CD81 is an entry coreceptor for hepatitis C virus.

Hepatitis C virus (HCV) envelope glycoproteins E1/E2 can pseudotype retroviral particles and efficiently mediate entry into target cells. Using this experimental system, we determined HCV tropism for different cell types. Only primary hepatocytes and one hepatoma cell line were susceptible to HCV pseudovirus entry, which could be inhibited by sera from HCV-infected individuals. Furthermore, expression of the putative HCV receptor CD81 on nonpermissive human hepatic but not murine cells enabled HCV pseudovirus entry. Importantly, inhibition of viral entry by an anti-CD81 mAb occurred at a step following HCV attachment to target cells. Our results indicate that CD81 functions as a post-attachment entry coreceptor and that other cellular factors act in concert with CD81 to mediate HCV binding and entry into hepatocytes.

L-SIGN (CD 209L) is a liver-specific capture receptor for hepatitis C virus.

Hepatitis C virus (HCV) infects nearly 3% of the population of the world and is a major cause of liver disease. However, the mechanism whereby the virus targets the liver for infection remains unknown, because none of the putative cellular receptors for HCV are both expressed specifically in the liver and capable of binding HCV envelope glycoproteins. Liver/lymph node-specific intercellular adhesion molecule-3-grabbing integrin (L-SIGN) is a calcium-dependent lectin expressed on endothelial cells of liver and lymph nodes. Dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN), a homologous molecule expressed on dendritic cells, binds HIV and promotes infection. By using a virus-binding assay, we demonstrate that L-SIGN and DC-SIGN specifically bind naturally occurring HCV present in the sera of infected individuals. Further studies demonstrate that binding is mediated by the HCV envelope glycoprotein E2 and is blocked by specific inhibitors, including mannan, calcium chelators, and Abs to the lectin domain of the SIGN molecules. Thus, L-SIGN represents a liver-specific receptor for HCV, and L-SIGN and DC-SIGN may play important roles in HCV infection and immunity.

L-SIGN (CD209L) and DC-SIGN (CD209) mediate transinfection of liver cells by hepatitis C virus.

Target cell tropism of enveloped viruses is regulated by interactions between viral and cellular factors during transmission, dissemination, and replication within the host. Binding of viral envelope glycoproteins to specific cell-surface receptors determines susceptibility to viral entry. However, a number of cell-surface molecules bind viral envelope glycoproteins without mediating entry. Instead, they serve as capture receptors that disseminate viral particles to target organs or susceptible cells. We and others recently demonstrated that the C type lectins L-SIGN and DC-SIGN capture hepatitis C virus (HCV) by specific binding to envelope glycoprotein E2. In this study, we use an entry assay to demonstrate that HCV pseudoviruses captured by L-SIGN+ or DC-SIGN+ cells efficiently transinfect adjacent human liver cells. Virus capture and transinfection require internalization of the SIGN-HCV pseudovirus complex. In vivo, L-SIGN is largely expressed on endothelial cells in liver sinusoids, whereas DC-SIGN is expressed on dendritic cells. Capture of circulating HCV particles by these SIGN+ cells may facilitate virus infection of proximal hepatocytes and lymphocyte subpopulations and may be essential for the establishment of persistent infection.