Validation of a high performance liquid chromatography method for quantitation of foot-and-mouth disease virus antigen in vaccines and vaccine manufacturing.

Foot-and-mouth disease (FMD) is an infectious viral disease that affects the main meat and dairy production animals, including cattle, sheep, goats and swine. It is readily transmissible and countries where the disease is present suffer harsh international trade restrictions on livestock products and serious economic losses. Vaccines are important tools to contain outbreaks and maintain the status of free with or without vaccination, as defined by the World Organization for Animal Health (OIE). The efficacy of vaccines is reliant on the content and integrity of inactivated virus particles. The long-established method to quantify the viral content of vaccines along the manufacturing process and in the final product is the 140S sucrose density gradient analysis. This method has been a valuable tool for many decades. However, it requires gradient preparation for each sample, a lengthy ultracentrifugation and a manual UV reading of the gradient, rendering it highly operator dependent and almost impossible to automate. We present a method to quantify FMDV particles in vaccines and intermediate process samples that is based on separation of components by size exclusion high performance liquid chromatography (SE-HPLC) and measurement of virus by absorption at 254 nm. The method has been extensively validated; it is accurate, precise and linear. It is applicable to all FMDV strains and sample materials and has a good concordance with the 140S test. The proposed method uses off the shelf HPLC equipment and columns. It is easily automated for high throughput operation, affording a useful process analytical technology and a novel tool for control of final product by manufacturers and regulatory agencies.

Foot and mouth disease (FMD) virus: quantification of whole virus particles during the vaccine manufacturing process by size exclusion chromatography.

Foot and mouth disease (FMD) is a highly infectious viral disease that affects cattle, sheep, goats and swine causing severe economic losses worldwide. The efficacy of inactivated vaccines is critically dependent on the integrity of foot and mouth disease virus (FMDV) particles. The recommended method to quantify the active ingredient of vaccines is the 140S quantitative sucrose density gradient analysis. This method has been an immensely valuable tool over the past three decades but it is highly operator dependent and difficult to automate. We developed a method to quantify FMDV particles during the vaccine manufacturing process that is based on separation of components by size-exclusion chromatography and measurement of virus by absorption at 254nm. The method is linear in the 5-70µg/mL range, it is applicable to different FMDV strains, and has a good correlation with the 140S test. The proposed method uses standard chromatographic media and it is amenable to automation. The method has potential as a process analytical technology and for control of final product by manufacturers, international vaccine banks and regulatory agencies.

Persistent anti-gag, -Nef, and -Rev IgM levels as markers of the impaired functions of CD4+ T-helper lymphocytes during SIVmac251 infection of cynomolgus macaques.

This study analyzed the antigen-specific (Gag, Nef, Rev, and Tat) IgM, IgG, and IgA humoral responses during the first 200 days of SIVmac251 infection in cynomolgus macaques. These responses were tested for correlation with the CD4(+) T-cell-related hematologic parameters and viral load throughout the course of the study (acute and chronic infection, during and after antiretroviral therapy). Strong inverse correlations were observed between the percentage of CD4(+) T cells at almost every timepoint of the study and the levels of IgM (but not IgG and IgA) against Gag, Nef, and Rev (but not Tat) measured after, but not during, the primary peak of IgM response. Significant levels of persistent antigen-specific IgMs may reflect the prevalence of mature plasma cells that have not undergone immunoglobulin class switching, possibly due to defects in helper T-cell function. Strong correlations were observed between the preinfection CD4(+) T-cell count or CD4/CD8 ratio and the same parameters measured throughout the study, suggesting the importance of preinfection immune status as a determinant of disease progression. The negative correlations between the post-acute-phase IgM levels and the percentage of CD4(+) T cells at later times during the study suggest the potential prognostic value of this measurement.

Analysis of IGG and IGG4 in HIV-1 seropositive patients and correlation with biological and genetic markers.

We have compared the levels of immunoglobulins G (IgG) and G4 (IgG4) in extreme seropositive patients from the GRIV cohort consisting of 168 patients with slow progression (SP) and 60 with rapid progression (RP) as well as in 173 healthy controls. IgG levels were significantly higher in SP patients than in RP patients (P = 0.008), both higher than in seronegative individuals. IgG4 levels were significantly lower in SP patients than in RP patients (P = 0.001), both lower than in seronegative individuals. We tried to correlate these levels with biological parameters (CD4(+) and CD8(+) cells, total lymphocytes, white blood cell counts, percentage of CD4(+) cells, and viral load) as well as with genetic markers from Th1/Th2 cytokines (IL2, IL4, IL6, IL10, IL13, and IFNgamma). IgG levels were correlated with the percentage of CD4(+) cells in SP while IgG4 levels were correlated with CD8(+) cell count in SP and with percentage of CD4(+) cells in RP patients. Among the parameters measured in SP patients at the time of inclusion in the study, the best predictor of progression towards AIDS was the viral load, the best predictor for stability was CD4(+) cell count, but overall, the best predictor for SP evolution (stability vs. progression) appeared to be the percentage of CD4(+) cells. Interestingly, correlations between the levels of IgG or IgG4 and the cytokine gene polymorphisms were found, notably in the IL10 gene.

T-cell and neuronal apoptosis in HIV infection: implications for therapeutic intervention.

The pathogenesis of HIV infection involves the selective loss of CD4+ T cells contributing to immune deficiency. Although loss of T cells leading to immune dysfunction in HIV infection is mediated in part by viral infection, there is a much larger effect on noninfected T cells undergoing apoptosis in response to activation stimuli. In the subset of patients with HIV dementia complex, neuronal injury, loss, and apoptosis are observed. Viral proteins, gp120 and Tat, exhibit proapoptotic activities when applied to T cell and neuronal cultures by direct and indirect mechanisms. The pathways leading to cell death involve the activation of one or more death receptor pathways (i.e., TNF-alpha, Fas, and TRAIL receptors), chemokine receptor signaling, cytokine dysregulation, caspase activation, calcium mobilization, and loss of mitochondrial membrane potential. In this review, the mechanisms involved in T-cell and neuronal apoptosis, as well as antiapoptotic pathways potentially amenable to therapeutic application, are discussed.

Immunogenicity of HIV-1 IIIB and SHIV 89.6P Tat and Tat toxoids in rhesus macaques: induction of humoral and cellular immune responses.

This study compared immune responses in rhesus macaques immunized with unmodified HIV-1 IIIB Tat, SHIV89.6P Tat, and carboxymethylated IIIB and 89.6P Tat toxoids. Immunization with either IIIB or 89.6P preparation induced high titer and broadly crossreactive serum anti-Tat IgG that recognized HIV-1 subtype-E and SIVmac251 Tat. However, the response was delayed, and titers were lower in 89.6P vaccination groups. Serum anti-Tat IgG recognized peptides corresponding to the amino-terminus, basic domain, and carboxy-terminal region. Cellular proliferative responses to Tat toxoids corresponding to the immunogen were evident in vitro in both IIIB and 89.6P groups. Crossreactive proliferative responses were observed in IIIB groups in response to stimulation with 89.6P or SIVmac251 Tat toxoids, but were much less prevalent in 89.6P groups. The truncated 86 amino acid IIIB Tat appears to be more immunogenic than the 102 amino acid 89.6P Tat with respect to both humoral and cellular immune responses, and may be a better vaccine component. Despite induction of robust humoral and cellular immune responses (including both CD4+ and CD8+ T-cell responses) to Tat, all animals were infected upon intravenous challenge with 30 MID(50) of SHIV89.6P and outcome of vaccine groups was not different from controls. Sequencing both Tat exons from serum viral RNA revealed no evidence of escape mutants. These results suggest that with intravenous SHIV89.6P challenge in rhesus macaques, precipitous CD4+ T-cell decline overwhelms potentially protective immune responses. Alternatively, Tat specific CD8+ T-cell responses may not appropriately recognize infected cells in vivo in this model. In view of evidence demonstrating Tat specific CTLs in the SIV model and in humans infected with HIV-1, results in this pathogenic SHIV model may not apparently predict the efficacy of this approach in human studies. The potency and cross-reactivity of these immune responses confirm Tat toxoid as an excellent candidate vaccine component.