Presentation by a major histocompatibility complex class I molecule of nucleoprotein peptide expressed in two different genes of an influenza virus transfectant.

Abstract

SummaryMajor histocompatibility (MHC) class I glycoproteins are specialized to present to CD8 + T cells, peptides that originate from proteins synthesized within the cytoplasm. Conventional killed vaccines are unable to get into the cell cytoplasm and therefore fail to expand the CD8 + T cell population. We have created a novel influenza transfectant virus, R10, which carries an immunogenic peptide from the nucleoprotein (NP) of PK8 influenza virus in its hemagglutinin (HA) and another similar peptide in its HK influenza virus NP. The two peptides are both presented by H-2D b and bind with approximately equal affinity. They can compete with one another for binding to H-2D b. Yet in cells infected with K10, both peptides are presented efficiently enough to expand the respective cytotoxic T lymphocyte (CTL) precursors in vivo and to serve as targets for CTL lysis in vitro. It has been proposed that proteins bearing signal sequences may be processed by a transporter-independent pathway. To investigate this, we infected the transporter-deficient cell line RMA-S with the R10 virus to see if the NP peptide expressed by the HA would be presented. The result shows that even the presence of a signal peptide in the HA does not overcome the lack of a transporter function, suggesting that the presentation of both peptides is dependent on functional transporter proteins. Our data also suggest the feasibility of creating by genetic engineering, recombinant vaccines expressing multiple epitopes that can effectively stimulate a cellular immune response.

DNA Immunization with Minigenes: Low Frequency of Memory Cytotoxic T Lymphocytes and Inefficient Antiviral Protection Are Rectified by Ubiquitination

“…Plasmids containing the isolated minigenes (MG34) failed to consistently protect mice, as did a plasmid in which the minigene cassette had been embedded in a fulllength polypeptide (pWRG-MG34). The latter finding was particularly surprising because several studies have shown that embedded epitopes can be presented by MHC class I (12,13,29) and can induce protective CTL responses (13). The protective efficacy of the MG34 sequence was, however, restored by ubiquitination; 75% of mice survived, on both MHC backgrounds.…”

A decade after the generation of a negative-sense RNA virus from cloned cDNA – what have we learned?

“…Influenza A viruses are promising vector candidates (reviewed by Garcia-Sastre, 2000 ) because of the availability of 15 HA and 9 NA subtypes, as well as numerous antigenic variants, which would allow repeated immunization. Recombinant viruses expressing foreign polypeptides integrated into the NA stalk or the antigenic sites of HA have been generated (Li et al ., 1992 , 1993a , b ; Castrucci et al ., 1994 ; Muster et al ., 1994 , 1995 ; Isobe et al ., 1995 ; Murata et al ., 1996 ; Gilleland et al ., 1997 ; Walker et al ., 1997 ). Results demonstrating immune responses against the foreign peptides include expression of the V3 loop of HIV-1 gp120 protein (Li et al ., 1993a ), expression of an epitope from the HIV-1 gp41 ectodomain (Muster et al ., 1994 , 1995 ) and expression of a cytotoxic T lymphocyte-specific epitope of the lymphocytic choriomeningitis virus nucleoprotein (Castrucci et al , 1994 ).…”

Generation of influenza A virus from cloned cDNAs — historical perspective and outlook for the new millenium

“…To explore the utility of influenza viruses as a vaccine vector, different research groups generated transfectant viruses expressing foreign polypeptides [reviewed in 102]. Viruses were constructed that expressed short foreign polypeptides integrated into one of the viral glycoproteins [72,[103][104][105][106][107][108][109][110][111]. Examples include the introduction of a cytotoxic T lymphocyte-specific epitope of the LCMV nucleoprotein in the NA stalk [72], the introduction of the V3 loop of HIV-1 gp120 protein or a highly conserved epitope from the ectodomain of HIV-1 gp41 into the antigenic site B of A/WSN/33 HA [104,106,108], or the replacement of the antigenic site B of A/WSN/33 (H1N1) with the corresponding sequence of H3 HA [103].…”