Mitigation of T-cell dependent immunogenicity by reengineering factor VIIa analogue.

Vatreptacog alfa (VA), a recombinant activated human factor VII (rFVIIa) variant with 3 amino acid substitutions, was developed to provide increased procoagulant activity in hemophilia patients with inhibitors to factor VIII or factor IX. In phase 3 clinical trials, changes introduced during the bioengineering of VA resulted in the development of undesired anti-drug antibodies in some patients, leading to the termination of a potentially promising therapeutic protein product. Here, we use preclinical biomarkers associated with clinical immunogenicity to validate our deimmunization strategy applied to this bioengineered rFVIIa analog.

A deimmunised form of the ribotoxin, α-sarcin, lacking CD4+ T cell epitopes and its use as an immunotoxin warhead.

Fungal ribotoxins that block protein synthesis can be useful warheads in the context of a targeted immunotoxin. α-Sarcin is a small (17 kDa) fungal ribonuclease produced by Aspergillus giganteus that functions by catalytically cleaving a single phosphodiester bond in the sarcin-ricin loop of the large ribosomal subunit, thus making the ribosome unrecognisable to elongation factors and leading to inhibition of protein synthesis. Peptide mapping using an ex vivo human T cell assay determined that α-sarcin contained two T cell epitopes; one in the N-terminal 20 amino acids and the other in the C-terminal 20 amino acids.

Small amounts of sub-visible aggregates enhance the immunogenic potential of monoclonal antibody therapeutics.

Purpose: Determine the effect of minute quantities of sub-visible aggregates on the in vitro immunogenicity of clinically relevant protein therapeutics.

Methods: Monoclonal chimeric (rituximab) and humanized (trastuzumab) antibodies were subjected to fine-tuned stress conditions to achieve low levels (<3% of total protein) of sub-visible aggregates. The effect of stimulating human dendritic cells (DC) and CD4(+) T cells with the aggregates was measured in vitro using cytokine secretion, proliferation and confocal microscopy.

Therapeutic vaccination against the rhesus lymphocryptovirus EBNA-1 homologue, rhEBNA-1, elicits T cell responses to novel epitopes in rhesus macaques.

Epstein-Barr virus (EBV) is a vaccine/immunotherapy target due to its association with several human malignancies. EBNA-1 is an EBV protein consistently expressed in all EBV-associated cancers. Herein, EBNA-1-specific T cell epitopes were evaluated after AdC-rhEBNA-1 immunizations in chronically lymphocryptovirus-infected rhesus macaques, an EBV infection model.

An Epstein-Barr virus encoded inhibitor of Colony Stimulating Factor-1 signaling is an important determinant for acute and persistent EBV infection.

Acute Epstein-Barr virus (EBV) infection is the most common cause of Infectious Mononucleosis. Nearly all adult humans harbor life-long, persistent EBV infection which can lead to development of cancers including Hodgkin Lymphoma, Burkitt Lymphoma, nasopharyngeal carcinoma, gastric carcinoma, and lymphomas in immunosuppressed patients. BARF1 is an EBV replication-associated, secreted protein that blocks Colony Stimulating Factor 1 (CSF-1) signaling, an innate immunity pathway not targeted by any other virus species.

Persistent infection drives the development of CD8+ T cells specific for late lytic infection antigens in lymphocryptovirus-infected macaques and Epstein-Barr virus-infected humans.

We examined the CD8(+) T cell repertoire against lytic infection antigens in rhesus macaques persistently infected with the Epstein-Barr virus (EBV)-related lymphocryptovirus (rhLCV). CD8(+) T cells specific for late (L) antigens were detected at rates comparable to those for early antigens and were associated with increasing duration of infection. L antigen-specific CD8(+) T cells were also readily detected in adult, EBV-positive humans.

Antibodies to lytic infection proteins in lymphocryptovirus-infected rhesus macaques: a model for humoral immune responses to epstein-barr virus infection.

Humoral immune responses to rhesus lymphocryptovirus (rhLCV) lytic infection proteins were evaluated in the rhesus macaque animal model for Epstein-Barr virus (EBV) infection. We found a hierarchy of humoral responses to 14 rhLCV lytic infection proteins in naturally infected rhesus macaques, with (i) widespread and robust responses to four glycoproteins expressed as late proteins, (ii) frequent but less robust responses to a subset of early proteins, and (iii) low-level responses to immediate-early proteins. This hierarchy of humoral responses was similar to that reported for EBV-infected humans, with the notable exception of the response to rhBARF1.

Decreased EBNA-1-specific CD8+ T cells in patients with Epstein-Barr virus-associated nasopharyngeal carcinoma.

The Epstein-Barr virus (EBV) nuclear antigen-1 (EBNA-1) is potentially a universal target for immune recognition of EBV-infected normal or malignant cells. EBNA-1-specific CD8+ T-cell responses have been assessed against a few epitopes presented on a limited number of HLA class I alleles. We now assess CD8+ T-cell responses to a complete panel of EBNA-1 peptides in an HLA-characterized population.

Influence of translation efficiency of homologous viral proteins on the endogenous presentation of CD8+ T cell epitopes.

A significant proportion of endogenously processed CD8(+) T cell epitopes are derived from newly synthesized proteins and rapidly degrading polypeptides (RDPs). It has been hypothesized that the generation of rapidly degrading polypeptides and CD8(+) T cell epitopes from these RDP precursors may be influenced by the efficiency of protein translation. Here we address this hypothesis by using the Epstein-Barr virus-encoded nuclear antigen 1 protein (EBNA1), with or without its internal glycine-alanine repeat sequence (EBNA1 and EBNA1DeltaGA, respectively), which display distinct differences in translation efficiency.

The BZLF1 homolog of an Epstein-Barr-related gamma-herpesvirus is a frequent target of the CTL response in persistently infected rhesus macaques.

Although CD8(+) T lymphocytes targeting lytic infection proteins dominate the immune response to acute and persistent EBV infection, their role in immune control of EBV replication is not known. Rhesus lymphocryptovirus (rhLCV) is a gamma-herpesvirus closely related to EBV, which establishes persistent infection in rhesus macaques. In this study, we investigated cellular immune responses to the rhLCV BZLF1 (rhBZLF1) homolog in a cohort of rhLCV-seropositive rhesus macaques.

The CD8+ T-cell response to an Epstein-Barr virus-related gammaherpesvirus infecting rhesus macaques provides evidence for immune evasion by the EBNA-1 homologue.

Epstein-Barr virus (EBV) infection persists for life in humans, similar to other gammaherpesviruses in the same lymphocryptovirus (LCV) genus that naturally infect Old World nonhuman primates. The specific immune elements required for control of EBV infection and potential immune evasion strategies essential for persistent EBV infection are not well defined. We evaluated the cellular immune response to latent infection proteins in rhesus macaques with naturally and experimentally acquired rhesus LCV (rhLCV) infection.

Reduced prevalence of Epstein-Barr virus-related lymphocryptovirus infection in sera from a new world primate.

The recent discovery of an Epstein-Barr virus (EBV)-related lymphocryptovirus (LCV) naturally infecting common marmosets demonstrated that gamma-1 herpesviruses are not limited to human and Old World nonhuman primate hosts. We developed serologic assays to detect serum antibodies against lytic- and latent-infection marmoset LCV antigens in order to perform the first seroepidemiologic study of LCV infection in New World primates. In three different domestic colonies and in animals recently captured from the wild, we found that the seroprevalence of marmoset LCV infection was not as ubiquitous as with EBV or Old World LCV.

Poliovirus proteins induce membrane association of GTPase ADP-ribosylation factor.

Poliovirus infection results in the disintegration of intracellular membrane structures and formation of specific vesicles that serve as sites for replication of viral RNA. The mechanism of membrane rearrangement has not been clearly defined. Replication of poliovirus is sensitive to brefeldin A (BFA), a fungal metabolite known to prevent normal function of the ADP-ribosylation factor (ARF) family of small GTPases.

Membrane requirements for uridylylation of the poliovirus VPg protein and viral RNA synthesis in vitro.

Efficient translation of poliovirus (PV) RNA in uninfected HeLa cell extracts generates all of the viral proteins required to carry out viral RNA replication and encapsidation and to produce infectious virus in vitro. In infected cells, viral RNA replication occurs in ribonucleoprotein complexes associated with clusters of vesicles that are formed from preexisting intracellular organelles, which serve as a scaffold for the viral RNA replication complex. In this study, we have examined the role of membranes in viral RNA replication in vitro.

Isolation of enzymatically active replication complexes from feline calicivirus-infected cells.

A membranous fraction that could synthesize viral RNA in vitro in the presence of magnesium salt, ribonucleotides, and an ATP-regenerating system was isolated from feline calicivirus (FCV)-infected cells. The enzymatically active component of this fraction was designated FCV replication complexes (RCs), by analogy to other positive-strand RNA viruses. The newly synthesized RNA was characterized by Northern blot analysis, which demonstrated the production of both full-length (8.