A targeted liquid chromatography mass spectrometry method for routine monitoring of cell culture media components for bioprocess development.

The analysis of cell culture media (CCM) components is critical for understanding cell growth kinetics and overall product quality during biomanufacturing. Given the diverse physical and chemical nature of CCM compounds present at a wide range of concentrations, there is an increasing demand for single-platform analytical assays with exceptional specificity and sensitivity. This study presents a targeted LC-MS/MS method for the identification and quantitation of 110 CCM analytes is presented, where target metabolites are monitored over an 20-min gradient. The analyte panel constitutes amino acids, vitamins, organic acids, nucleic acids, carbohydrates, and lipids. The method employs isotopically labeled standards to enable specific and accurate relative quantitation of CCM compounds based on physicochemical properties and retention time. Quantitation is performed on a triple quadrupole mass spectrometer operated in multiple reaction monitoring (MRM) mode. The method demonstrates strong linearity with an R2 of ≥0.99 with three orders of linear dynamic range and inter-day and intra-day precision with a%CV of <10% for spiked-in quality control samples. We also present three case studies to demonstrate method applicability in the bioprocessing space for developing vaccines and biologics.

Immunogenicity and Efficacy of a Novel Multi-Antigenic Peptide Vaccine Based on Cross-Reactivity between Feline and Human Immunodeficiency Viruses.

For the development of an effective HIV-1 vaccine, evolutionarily conserved epitopes between feline and human immunodeficiency viruses (FIV and HIV-1) were determined by analyzing overlapping peptides from retroviral genomes that induced both anti-FIV/HIV T cell-immunity in the peripheral blood mononuclear cells from the FIV-vaccinated cats and the HIV-infected humans. The conserved T-cell epitopes on p24 and reverse transcriptase were selected based on their robust FIV/HIV-specific CD8⁺ cytotoxic T lymphocyte (CTL), CD4⁺ CTL, and polyfunctional T-cell activities. Four such evolutionarily conserved epitopes were formulated into four multiple antigen peptides (MAPs), mixed with an adjuvant, to be tested as FIV vaccine in cats. The immunogenicity and protective efficacy were evaluated against a pathogenic FIV. More MAP/peptide-specific CD4⁺ than CD8⁺ T-cell responses were initially observed. By post-third vaccination, half of the MAP/peptide-specific CD8⁺ T-cell responses were higher or equivalent to those of CD4⁺ T-cell responses. Upon challenge, 15/19 (78.9%) vaccinated cats were protected, whereas 6/16 (37.5%) control cats remained uninfected, resulting in a protection rate of 66.3% preventable fraction (p = 0.0180). Thus, the selection method used to identify the protective FIV peptides should be useful in identifying protective HIV-1 peptides needed for a highly protective HIV-1 vaccine in humans.

Conserved epitopes on HIV-1, FIV and SIV p24 proteins are recognized by HIV-1 infected subjects.

Cross-reactive peptides on HIV-1 and FIV p24 protein sequences were studied using peripheral blood mononuclear cells (PBMC) from untreated HIV-1-infected long-term survivors (LTS; >10 y of infection without antiretroviral therapy, ART), short-term HIV-1 infected subjects not on ART, and ART-treated HIV-1 infected subjects. IFNγ-ELISpot and CFSE-proliferation analyses were performed with PBMC using overlapping HIV-1 and FIV p24 peptides. Over half of the HIV-1 infected subjects tested (22/31 or 71%) responded to one or more FIV p24 peptide pools by either IFNγ or T-cell proliferation analysis. PBMC and T cells from infected subjects in all 3 HIV(+) groups predominantly recognized one FIV p24 peptide pool (Fp14) by IFNγ production and one additional FIV p24 peptide pool (Fp9) by T-cell proliferation analysis. Furthermore, evaluation of overlapping SIV p24 peptide sequences identified conserved epitope(s) on the Fp14/Hp15-counterpart of SIV, Sp14, but none on Fp9-counterpart of SIV, Sp9. The responses to these FIV peptide pools were highly reproducible and persisted throughout 2-4 y of monitoring. Intracellular staining analysis for cytotoxins and phenotyping for CD107a determined that peptide epitopes from Fp9 and Fp14 pools induced cytotoxic T lymphocyte-associated molecules including perforin, granzyme B, granzyme A, and/or expression of CD107a. Selected FIV and corresponding SIV epitopes recognized by HIV-1 infected patients indicate that these protein sequences are evolutionarily conserved on both SIV and HIV-1 (e.g., Hp15:Fp14:Sp14). These studies demonstrate that comparative immunogenicity analysis of HIV-1, FIV, and SIV can identify evolutionarily-conserved T cell-associated lentiviral epitopes, which could be used as a vaccine for prophylaxis or immunotherapy.

Evolutionarily conserved epitopes on human immunodeficiency virus type 1 (HIV-1) and feline immunodeficiency virus reverse transcriptases detected by HIV-1-infected subjects.

Anti-human immunodeficiency virus (HIV) cytotoxic T lymphocyte (CTL)-associated epitopes, evolutionarily conserved on both HIV type 1 (HIV-1) and feline immunodeficiency virus (FIV) reverse transcriptases (RT), were identified using gamma interferon (IFN-γ) enzyme-linked immunosorbent spot (ELISpot) and carboxyfluorescein diacetate succinimide ester (CFSE) proliferation assays followed by CTL-associated cytotoxin analysis. The peripheral blood mononuclear cells (PBMC) or T cells from HIV-1-seropositive (HIV(+)) subjects were stimulated with overlapping RT peptide pools. The PBMC from the HIV(+) subjects had more robust IFN-γ responses to the HIV-1 peptide pools than to the FIV peptide pools, except for peptide-pool F3. In contrast, much higher and more frequent CD8(+) T-cell proliferation responses were observed with the FIV peptide pools than with the HIV peptide pools. HIV-1-seronegative subjects had no proliferation or IFN-γ responses to the HIV and FIV peptide pools. A total of 24% (40 of 166) of the IFN-γ responses to HIV pools and 43% (23 of 53) of the CD8(+) T-cell proliferation responses also correlated to responses to their counterpart FIV pools. Thus, more evolutionarily conserved functional epitopes were identified by T-cell proliferation than by IFN-γ responses. In the HIV(+) subjects, peptide-pool F3, but not the HIV H3 counterpart, induced the most IFN-γ and proliferation responses. These reactions to peptide-pool F3 were highly reproducible and persisted over the 1 to 2 years of testing. All five individual peptides and epitopes of peptide-pool F3 induced IFN-γ and/or proliferation responses in addition to inducing CTL-associated cytotoxin responses (perforin, granzyme A, granzyme B). The epitopes inducing polyfunctional T-cell activities were highly conserved among human, simian, feline, and ungulate lentiviruses, which indicated that these epitopes are evolutionarily conserved. These results suggest that FIV peptides could be used in an HIV-1 vaccine.

HIV-1 Vaccine Trials: Evolving Concepts and Designs.

An effective prophylactic HIV-1 vaccine is needed to eradicate the HIV/AIDS pandemic but designing such a vaccine is a challenge. Despite many advances in vaccine technology and approaches to generate both humoral and cellular immune responses, major phase-II and -III vaccine trials against HIV/AIDS have resulted in only moderate successes. The modest achievement of the phase-III RV144 prime-boost trial in Thailand re-emphasized the importance of generating robust humoral and cellular responses against HIV. While antibody-directed approaches are being pursued by some groups, others are attempting to develop vaccines targeting cell-mediated immunity, since evidence show CTLs to be important for the control of HIV replication. Phase-I and -IIa multi-epitope vaccine trials have already been conducted with vaccine immunogens consisting of known CTL epitopes conserved across HIV subtypes, but have so far fallen short of inducing robust and consistent anti-HIV CTL responses. The concepts leading to the development of T-cell epitope-based vaccines, the outcomes of related clinical vaccine trials and efforts to enhance the immunogenicity of cell-mediated approaches are summarized in this review. Moreover, we describe a novel approach based on the identification of SIV and FIV antigens which contain conserved HIV-specific T-cell epitopes and represent an alternative method for developing an effective HIV vaccine against global HIV isolates.

Feline immunodeficiency virus model for designing HIV/AIDS vaccines.

Feline immunodeficiency virus (FIV) discovered in 1986 is a lentivirus that causes AIDS in domestic cats. FIV is classified into five subtypes (A-E), and all subtypes and circulating intersubtype recombinants have been identified throughout the world. A commercial FIV vaccine, consisting of inactivated subtype-A and -D viruses (Fel-O-Vax FIV, Fort Dodge Animal Health), was released in the United States in 2002. The United States Department of Agriculture approved the commercial release of Fel-O-Vax FIV based on two efficacy trials using 105 laboratory cats and a major safety trial performed on 689 pet cats. The prototype and commercial FIV vaccines had broad prophylactic efficacy against global FIV subtypes and circulating intersubtype recombinants. The mechanisms of cross-subtype efficacy are attributed to FIV-specific T-cell immunity. Findings from these studies are being used to define the prophylactic epitopes needed for an HIV-1 vaccine for humans.