High level expression and secretion of truncated forms of herpes simplex virus type 1 and type 2 glycoprotein D by the methylotrophic yeast Pichia pastoris.

Herpes simplex virus type 1 and 2 (HSV-1 and -2) glycoproteins D (gD-1 and gD-2) play a role in the entry of the virus into the host cell. Availability of substantial amounts of these proteins, or large fragments thereof, will be needed to allow studies at the molecular level. We studied the potency of the Pichia pastoris yeast expression system to produce soluble forms of gD. The DNA sequences encoding the extracellular domains of gD [amino acids 1-314 (gD-1(1-314)) and amino acids 1-254 (gD-1(1-254)) of gD-1 and amino acids 1-314 of gD-2 (gD-2(1-314))] were cloned into the P. pastoris yeast expression vector pPIC9. Two truncated forms of gD-1 were fitted with a His tail (designated as gD-1(1-314His) and gD-1(1-254His)) to facilitate their purification. Large amounts of gD-1(1-314) and gD-1(1-314His) (280-300mg/L induction medium) were produced. The yields of recombinant gD-1(1-254) and gD-1(1-254His) were lower: 20-36mg/L, and the yield of the gD-2(1-314) fragment was much lower: 6mg/L. SDS-PAGE analysis revealed multiple glycosylated species of the larger gD fragments, ranging in apparent molecular weight from 31 to 78kDa. The smaller gD-1(1-254) fragment appeared as two bands with molecular weights of 33 and 31kDa. All recombinant proteins produced by P. pastoris were recognized, as expected, by a panel of MAbs (A16, DL6, A18, DL11, HD1, ABDI, and AP7). In addition, we showed that gD-1(1-314), gD-2(1-314), and gD-1(1-254His) were able to interfere with binding of HSV to susceptible cells. These results indicate that the conformations of the recombinant proteins closely resemble those of native gD.

Natural infection with herpes simplex virus type 1 (HSV-1) induces humoral and T cell responses to the HSV-1 glycoprotein H:L complex.

The glycoproteins of herpes simplex virus type 1 (HSV-1) are important targets for the immune system in the control of HSV-1 infections. The humoral and T cell responses to the glycoprotein (g)H(t(His)):gL complex of HSV-1 were studied in seven HSV-1-seropositive and three HSV-1-seronegative healthy adults. In addition, responses to HSV-1 gD(t) were determined. As antigens, purified soluble recombinant forms of the gH(t(His)):gL complex produced by insect cells and of gD(t) produced by yeast cells were used. In contrast to seronegative donors, sera of all seropositive donors contained gH(t(His)): gL-specific IgG. Using peripheral blood (PB) T cells, gH(t(His)):gL-specific proliferative T cell responses were detected in all seropositive donors. Culture supernatants of PB T cells stimulated with recombinant gH(t(His)):gL contained high levels of interferon-gamma and no detectable interleukin-4, indicating their Th1 phenotype. These results show that naturally acquired HSV-1 infection induces gH:gL-specific humoral and T cell responses.