DNA shuffling and vaccines.

One challenge of biotechnology is to find ways to optimize enzymes, cytokines, vaccines or transgenes in new contexts that are typically not found in nature. The approach of DNA shuffling is a test-tube process that takes advantage of recombination to generate libraries of chimeric genes, which can then be screened to identify the encoded proteins improved in one or more functions. DNA shuffling of two or more genes that are structurally similar and homologous in function is particularly efficacious in generating large libraries of functionally novel molecules. Other directed evolution methods, such as those involving directed or random mutagenesis, have several limitations compared to the DNA shuffling recombination process. A wide variety of genes have been submitted to DNA shuffling, and significant improvements in various functional parameters have been obtained. Several examples of the application of DNA shuffling to vaccine development, therapeutics and gene therapy are discussed here.

Evaluation of a live attenuated recombinant virus RAV 9395 as a herpes simplex virus type 2 vaccine in guinea pigs.

Recombinant virus RAV 9395 was constructed by deleting both copies of the gamma(1)34.5 gene, and the UL55 and UL56 open reading frames from herpes simplex virus type 2 (HSV-2) strain G. The potential use of RAV 9395 as an HSV-2 vaccine was investigated by evaluating the ability of RAV 9395 to protect guinea pigs from severe disease by HSV-2(G) challenge. RAV 9395 administered intramuscularly reduced both lesion development and severity in a dose-dependent manner in guinea pigs challenged with HSV-2(G). The frequency of reactivation of RAV 9395 from explanted dorsal root ganglia was low compared with that of HSV-2(G). Immunization with RAV 9395 at doses of 1 x 10(5) pfu and above generally precluded the establishment of latency by the challenge virus. The results presented in this report lend support for the development of genetically engineered live HSV vaccines.

An avirulent ICP34.5 deletion mutant of herpes simplex virus type 1 is capable of in vivo spontaneous reactivation.

The herpes simplex virus type 1 (HSV-1) ICP34.5 gene is a neurovirulence gene in mice. In addition, some ICP34.5 mutants have been reported to have a reduced efficiency of induced reactivation as measured by in vitro explantation of latently infected mouse ganglia. However, since spontaneous reactivation is almost nonexistent in mice, nothing has been reported on the effect of ICP34.5 mutants on spontaneous reactivation in vivo. To examine this, we have deleted both copies of the ICP34.5 neurovirulence gene from a strain of HSV-1 (McKrae) that has a high spontaneous reactivation rate in rabbits and used this mutant to infect rabbit eyes. All rabbits infected with the ICP34.5 mutant virus (d34.5) survived, even at challenge doses greater than 4 x 10(7) PFU per eye. In contrast, a 200-fold-lower challenge dose of 2 x 10(5) PFU per eye was lethal for approximately 50% of rabbits infected with either the wild-type McKrae parental virus or a rescued ICP34.5 mutant in which both copies of the ICP34.5 gene were restored. In mice, the 50% lethal dose of the ICP34.5 mutant was over 10(6) PFU, compared with a value of less than 10 PFU for the rescued virus. The ICP34.5 mutant was restricted for replication in rabbit and mouse eyes and mouse trigeminal ganglia in vivo. The spontaneous reactivation rate in rabbits for the mutant was 1.4% as determined by culturing tear films for the presence of reactivated virus. This was more than 10-fold lower than the spontaneous reactivation rate determined for the rescued virus (19.6%) and was highly significant (P < 0.0001, Fisher exact test). Southern analysis confirmed that the reactivated virus retained both copies of the ICP34.5 deletion. Thus, this report demonstrates that (i) the ICP34.5 gene, known to be a neurovirulence gene in mice, is also important for virulence in rabbits and (ii) in vivo spontaneous reactivation of HSV-1 in the rabbit ocular model, although reduced, can occur in the absence of the ICP34.5 gene.

Local expression of tumor necrosis factor alpha and interleukin-2 correlates with protection against corneal scarring after ocular challenge of vaccinated mice with herpes simplex virus type 1.

To correlate specific local immune responses with protection from corneal scarring, we examined immune cell infiltrates in the cornea after ocular challenge of vaccinated mice with herpes simplex virus type 1 (HSV-1). This is the first report to examine corneal infiltrates following ocular challenge of a vaccinated mouse rather than following infection of a naive mouse. Mice were vaccinated systemically with vaccines that following ocular challenge with HSV-1 resulted in (i) complete protection against corneal disease (KOS, an avirulent strain of HSV-1); (ii) partial protection, resulting in moderate corneal disease (baculovirus-expressed HSV-1 glycoprotein E [gE]); and (iii) no protection, resulting in severe corneal disease (mock vaccine). Infiltration into the cornea of CD4+ T cells, CD8+ T cells, macrophages, and cells containing various lymphokines was monitored on days 0, 1, 3, 7, and 10 postchallenge by immunocytochemistry of corneal sections. Prior to ocular challenge, no eye disease or corneal infiltrates were detected in any mice. KOS-vaccinated mice developed high HSV-1 neutralizing antibody titers (> 1:640) in serum. After ocular challenge, they were completely protected against death, developed no corneal disease, and had no detectable virus in their tear films at any time examined. In response to the ocular challenge, these mice developed high local levels of infiltrating CD4+ T cells and cells containing interleukin-2 (IL-2), IL-4, IL-6, or tumor necrosis factor alpha (TNF-alpha). In contrast, only low levels of infiltrating CD8+ T cells were found, and gamma interferon (IFN-gamma)-containing cells were not present until day 10. gE-vaccinated mice developed neutralizing antibody titers in serum almost as high as those of the KOS-vaccinated mice (> 1:320). After ocular challenge, they were also completely protected against death. However, the gE-vaccinated mice developed low levels of corneal disease and virus was detected in one-third of their eyes. Compared with KOS-vaccinated mice, the gE-vaccinated mice had a similar pattern of IFN-gamma, but a delay in the appearance of CD4+ T cells, CD8+ T cells, and IL-4-, IL-6-, and TNF-alpha-containing cells. In sharp contrast to those of the KOS-vaccinated mice, no cells containing IL-2 were detected in the eyes of gE-vaccinated mice at any time. Mock-vaccinated mice developed no detectable neutralizing antibody titer and were not protected from lethal HSV-1 challenge.(ABSTRACT TRUNCATED AT 400 WORDS)

The latency-associated transcript gene of herpes simplex virus type 1 (HSV-1) is required for efficient in vivo spontaneous reactivation of HSV-1 from latency.

During herpes simplex virus type 1 (HSV-1) neuronal latency, the only viral RNA detected is from the latency-associated transcript (LAT) gene. We have made a LAT deletion mutant of McKrae, an HSV-1 strain with a very high in vivo spontaneous reactivation rate. This mutant (dLAT2903) lacks the LAT promoter and the first 1.6 kb of the 5' end of LAT. dLAT2903 was compared with its parental virus and with a rescued virus containing a restored LAT gene (dLAT2903R). Replication of the LAT mutant in tissue culture, rabbit eyes, and rabbit trigeminal ganglia was similar to that of the rescued and parental viruses. On the basis of semiquantitative PCR analysis of the amount of HSV-1 DNA in trigeminal ganglia, the LAT mutant was unimpaired in its ability to establish latency. In contrast, spontaneous reactivation of dLAT2903 in the rabbit ocular model of HSV-1 latency and reactivation was decreased to approximately 33% of normal. This decrease was highly significant (P < 0.0001) and demonstrates that in an HSV-1 strain with a high spontaneous reactivation rate, deletion of LAT can dramatically decrease in vivo spontaneous reactivation. We also report here that deletion of LAT appeared to eliminate rather than just reduce in vivo induced reactivation.

Similarities in regulation of the HSV-1 LAT promoter in corneal and neuronal cells.

PURPOSE: To address the possibility of neuronal-like herpes simplex virus type 1 (HSV-1) latency in the cornea by determining if regulation of the HSV-1 LAT promoter in stromal keratocytes is similar to LAT promoter regulation in neurons. METHODS: Transient chloramphenicol acetyltransferase (CAT) assays were used to measure the relative promoter activity of various HSV-1 LAT promoter fragments in primary human corneal cells versus neuronal and nonneuronal cells. RESULTS: The authors found that the LAT promoter, whose location they previously mapped in neurons using transient CAT assays, functioned in stromal keratocytes using the same assay system and that two regions between -283 and -1932 nucleotides relative (upstream) to the start of LAT transcription slightly increased the LAT promoter activity in stromal keratocytes. They previously showed a similar increase in neuronal cells, and a large decrease in nonneuronal cells. In addition, they found that a neuronal specific enhancer region they previously defined between -162 and -283 nucleotides upstream of the start of LAT transcription also enhanced promoter activity in stromal keratocytes. Using gel-shift assays, they detected a nuclear factor specific to neurons and stromal keratocytes that binds to the LAT promoter and that may be a LAT regulatory factor. CONCLUSIONS: Recently, it has been suggested that the cornea might serve as an alternative site of latent herpes simplex virus type 1 (HSV-1) infection. However, this remains controversial. The authors' findings suggest that corneal and neuronal cells regulate the LAT promoter similarly and that this regulation differs from that seen in nonneuronal cells. Thus, the possibility of neuronal-like latency in the cornea remains plausible.

Expression of seven herpes simplex virus type 1 glycoproteins (gB, gC, gD, gE, gG, gH, and gI): comparative protection against lethal challenge in mice.

We have constructed recombinant baculoviruses individually expressing seven of the herpes simplex virus type 1 (HSV-1) glycoproteins (gB, gC, gD, gE, gG, gH, and gI). Vaccination of mice with gB, gC, gD, gE, or gI resulted in production of high neutralizing antibody titers to HSV-1 and protection against intraperitoneal and ocular challenge with lethal doses of HSV-1. This protection was statistically significant and similar to the protection provided by vaccination with live nonvirulent HSV-1 (90 to 100% survival). In contrast, vaccination with gH produced low neutralizing antibody titers and no protection against lethal HSV-1 challenge. Vaccination with gG produced no significant neutralizing antibody titer and no protection against ocular challenge. However, gG did provide modest, but statistically significant, protection against lethal intraperitoneal challenge (75% protection). Compared with the other glycoproteins, gG and gH were also inefficient in preventing the establishment of latency. Delayed-type hypersensitivity responses to HSV-1 at day 3 were highest in gG-, gH-, and gE-vaccinated mice, while on day 6 mice vaccinated with gC, gE, and gI had the highest delayed-type hypersensitivity responses. All seven glycoproteins produced lymphocyte proliferation responses, with the highest response being seen with gG. The same five glycoproteins (gB, gC, gD, gE, and gI) that induced the highest neutralization titers and protection against lethal challenge also induced some killer cell activity. The results reported here therefore suggest that in the mouse protection against lethal HSV-1 challenge and the establishment of latency correlate best with high preexisting neutralizing antibody titers, although there may also be a correlation with killer cell activity.

An improved method for cloning portions of the repeat regions of herpes simplex virus type 1.

The use of a low copy number plasmid to enhance greatly the ability to clone herpes simplex virus type 1 (HSV-1) DNA is described. Certain regions of the HSV-1 DNA have been extremely difficult to clone. Particular difficulties have often been encountered in the long and short viral repeats. Attempts to clone certain HSV-1 sequences using common plasmids produced plasmids containing inserts of unusual size and/or plasmids smaller than the original plasmid. The reason for these cloning difficulties is unclear. However, the difficulties may be related to the high overall GC content of the HSV-1 genome, the even higher GC content of the repeats, small direct and inverted repeats within the viral repeats, or uncharacterized secondary DNA structure. We show that the use of the low copy number plasmid pEV-vrf3 greatly enhanced our ability to clone and subclone 'difficult' HSV-1 restriction fragments, including restriction fragments that we were unable to clone using other plasmids.

Expression and characterization of baculovirus expressed herpes simplex virus type 1 glycoprotein L.

We have constructed a recombinant baculovirus expressing high levels of the herpes simplex virus type 1 (HSV-1) glycoprotein L (gL) in Sf9 cells. Sf9 cells infected with this recombinant virus synthesized three polypeptides of 26-27 kDa 28 kDa, and 31 kDa. The 28 and 31 kDa species were sensitive to tunicamycin and N-glycosidase F (PNGase F) treatment, suggesting that they were glycosylated. As shown by both indirect immunofluorescence and Western blot analysis, using polyclonal antibodies to synthetic gL peptides indicated that the baculovirus expressed gL was abundant on the surface of baculovirus gL infected Sf9 cells. A small fraction of the 31 kDa polypeptide was secreted into the extracellular medium as judged by Western blot analysis. The secreted form of gL was completely resistant to Endoglycosidase H (Endo-H), while the membrane associated form of gL was only partially resistant to Endo-H treatment, suggesting that the secreted gL represented a subpopulation of the membrane bound gL. Mice vaccinated with baculovirus expressed gL produced serum antibodies that reacted with authentic HSV-1 gL. However, these mice produced no HSV-1 neutralizing antibody (titer < 1:10) and they were not protected from lethal intraperitoneal or lethal ocular challenge with HSV-1. Thus, when used as a vaccine in the mouse model, gL, similar to our findings with HSV-1 gH, but unlike our results with the other 6 HSV-1 glycoproteins that we have expressed in this baculovirus system, did not provide any protection against HSV-1 challenge.

Baculovirus-expressed glycoprotein G of herpes simplex virus type 1 partially protects vaccinated mice against lethal HSV-1 challenge.

The DNA sequence encoding the complete HSV-1 glycoprotein G (gG) was inserted into a baculovirus transfer vector and recombinant viruses expressing gG were isolated. Three gG-related recombinant baculovirus expressed peptides of 37, 42, and 44 kDa were detected by Western blotting using monoclonal antibody to gG. The 42- and 44-kDa species were susceptible to tunicamycin, Endoglycosidase H (Endo-H), and N-glycosidase F (PNGase F) treatments, suggesting that they were glycosylated. Although only very low levels (approximately 1:10) of HSV-1-neutralizing antibody were produced in mice vaccinated with the baculovirus gG, these mice were partially protected from lethal challenge with HSV-1 (75-78% survival) and this level of protection was highly significant (P = 0.002). This is the first report to show that vaccination with HSV-1 gG can provide mice with any level of protection against lethal HSV-1 challenge.

Baculovirus expressed herpes simplex virus type 1 glycoprotein C protects mice from lethal HSV-1 infection.

A recombinant baculovirus (vAc-gC1) was constructed that expresses the glycoprotein C (gC) gene of herpes simplex virus type 1 (HSV-1). When Sf9 cells were infected with this recombinant, a protein that was smaller in size than authentic HSV-1 gC was detected by Western blotting using anti-gC polyclonal antibody. The recombinant gC was susceptible to tunicamycin, partially resistant to Endo-H, and was found on the membrane of Sf9 cells. Antibodies raised in mice to recombinant gC reacted with gC from HSV-1 infected cells and neutralized the infectivity of HSV-1 in vitro. Immunized mice were protected from lethal challenge with HSV-1.

Baculovirus-expressed glycoprotein E (gE) of herpes simplex virus type-1 (HSV-1) protects mice against lethal intraperitoneal and lethal ocular HSV-1 challenge.

We have constructed a recombinant baculovirus expressing high levels of the herpes simplex virus type 1 (HSV-1) glycoprotein E (gE) in Sf9 cells. The expressed gE migrated on gels as a double band with apparent molecular weights of 68 and 70 kDa. The recombinant gE was glycosylated based on its susceptibility to tunicamycin treatment and was transported to the membrane of Sf9 cells based on indirect immunofluorescence. Mice vaccinated with gE developed high serum titers of HSV-1-neutralizing antibodies based on plaque reduction assays. gE vaccination also induced a strong delayed type hypersensitivity (DTH) response to HSV-1. In addition, mice vaccinated with the recombinant gE were protected from both intraperitoneal and ocular lethal HSV-1 challenge. To our knowledge, this is the first report in which vaccination with gE was shown to induce high neutralizing antibody titers, a DTH response, or protection against lethal HSV-1 challenge.

Expression of herpes simplex virus type 1 glycoprotein I in baculovirus: preliminary biochemical characterization and protection studies.

We have constructed a recombinant baculovirus expressing the herpes simplex virus type 1 (HSV-1) glycoprotein I (gI). Sf9 cells infected with this recombinant virus synthesized gI-related polypeptides with apparent molecular sizes of 52 and 56 kDa. The recombinant gI appeared to be glycosylated, since it was susceptible to both tunicamycin and endoglycosidase H, and the expressed gI was transported to the surface of infected cells as judged by indirect immunofluorescence. Antibodies to the recombinant gI raised in mice neutralized HSV-1 infectivity. Finally, we show here for the first time that vaccination with gI can protect mice against HSV-1 challenge.

Baculovirus-expressed glycoprotein H of herpes simplex virus type 1 (HSV-1) induces neutralizing antibody and delayed type hypersensitivity responses, but does not protect immunized mice against lethal HSV-1 challenge.

We have shown previously that herpes simplex virus type 1 (HSV-1) glycoprotein H (gH) expressed by a baculovirus recombinant is transported to the cell surface in the absence of other HSV-1 gene products, and that the expressed gH has an apparent Mr similar to that of authentic HSV-1 gH. We report here that antibodies raised in mice to this baculovirus-expressed gH neutralize the infectivity of HSV-1 in vitro; this neutralizing activity was not complement-dependent. Mice vaccinated with gH also developed delayed type hypersensitivity (DTH) to HSV-1. This is the first report of expressed HSV-1 gH inducing neutralizing antibody or DTH responses in vaccinated animals. In contrast to the gH expressed in mammalian systems, the ability of this baculovirus-expressed gH to induce a neutralizing antibody response may be due to the inability of the mammalian expression system to transport gH to the cell surface. Despite inducing anti-HSV-1 neutralizing antibody and DTH responses, vaccination of mice with gH did not protect the mice against lethal intraperitoneal challenge with HSV-1.

Expression of herpes simplex virus type 1 glycoprotein B in insect cells. Initial analysis of its biochemical and immunological properties.

A recombinant baculovirus (vAc-gB1) was constructed which expresses the glycoprotein B (gB) gene of herpes simplex virus type 1 (HSV-1). When Sf9 cells were infected with these recombinant viruses, a protein that was close in size to authentic HSV-1 gB was detected by gB polyclonal antibody. The recombinant gB was found on the membrane of Sf9 cells and was susceptible to tunicamycin, glycosidase F (PNGase F) and partially susceptible to Endo-H. Antibodies raised in mice to this recombinant recognized viral gB and neutralized the infectivity of HSV-1 in vitro. Mice inoculated with the recombinant gB were protected from lethal challenge with HSV-1.

Immunoselection of recombinant baculoviruses expressing high levels of biologically active herpes simplex virus type 1 glycoprotein D.

The DNA sequence encoding the complete herpes simplex virus type 1 (HSV-1) glycoprotein D (gD) was inserted into a baculovirus transfer vector under control of the polyhedrin gene promoter of the baculovirus Autographa california nuclear polyhedrosis virus (AcNPV). After co-transfection of Spodoptera frugiperda (Sf9) insect cells with wild-type AcNPV DNA and the recombinant transfer vector DNA, polyhedrin-negative recombinants that expressed high levels of HSV-1 gD were isolated using immunoaffinity selection with antibody coated magnetic particles followed by plaque purification. These recombinant baculoviruses expressed a protein that was slightly smaller than virion HSV-1 gD made in Vero cells. This recombinant protein was expressed at high levels. The expressed protein was glycosylated, was found on the membrane of Sf9 cells, and reacted with gD specific antibodies. Antibodies raised in mice to the recombinant gD neutralized HSV-1 as measured by plaque reduction assays. Mice inoculated with the recombinant baculovirus were completely protected from lethal challenge with HSV-1.

An assay for the detection of interferon dependent and interferon independent antibody activities.

A spectrophotometric assay is described for the detection of interferon (IFN) dependent antibody (IDA) activity (i.e., antibodies that act with IFN to synergistically inhibit virus infection) and IFN independent antibody (IIA) activity (i.e., antibodies that act additively with IFN to inhibit virus infection). Four neutralizing monoclonal antibodies (mAb) against poliovirus type 1 (PV-1) were tested. Three mAb exhibited IDA activity and one mAb exhibited IIA activity against PV-1 strain Sabin. Concomitantly, the respective IDA and IIA activities were confirmed by a yield reduction assay. Also, the spectrophotometric assay detected IIA activity against PV-1 and IDA activity against PV-2 and PV-3 in human serum. Interestingly, antibody to a synthetic peptide of PV-1 capsid protein VP2 exhibited IIA activity against PV-2 strain MEF. Thus, this assay can facilitate the identification and investigation of IDA and IIA activities. Further, the assay adds economic feasibility to studying the natural occurrence of these antibody activities in the general population and can be useful in assessing the therapeutic potential of vaccine induced and hyperimmune antibodies.

Interferon-gamma can remain on the cell surface during the induction of the antiviral state.

The antiviral activity of cell-associated, non-elutable recombinant human gamma interferon (rHuIFN-gamma) was neutralized by antibody. The neutralization of cell-associated rHuIFN-gamma was maximal through 2 h (60-100%) and declined through 8 h (20-40%). Concomitantly, the antiviral activity of cell-associated [Met-Gln-Asp-Pro]-rHuIFN-gamma was sensitive to trypsin digestion over the same time period. However, the cell-associated antiviral activity of [Cys-Tyr-Cys]-rHuIFN-gamma remained sensitive to trypsin through 8 h. Neutralization of cell-associated rHuIFN-gamma by antibodies to the N-terminal end of HuIFN-gamma suggests that the N-terminal end(s) of cell-associated rHuIFN-gamma is directed outward from the receptor. Further, immunoprecipitation of radio-labelled rHuIFN-gamma by antibody alone suggests that biologically active rHuIFN-gamma is an oligomer. Taken together, these studies suggest that neutralization of cell-associated rHuIFN-gamma is probably due to divalent binding of antibody to or between rHuIFN-gamma in receptors on the cell surface. Also, our studies indicate that rHuIFN-gamma can remain associated with the cell surface during the induction of the antiviral state (AVS) and that binding of antibody to cell-associated rHuIFN-gamma inhibits the molecular events responsible for induction of the AVS.

Cytopathogenicity, drug susceptibility, and thymidine kinase activity of a retinovirulent herpes simplex virus type 2.

We investigated some of the biological and biochemical characteristics of a neuroinvasive, retinovirulent herpes simplex virus type 2 strain SL (HSV-2[SL]) and compared them with those of a neurovirulent, nonretinovirulent HSV-2 (186). HSV-2(SL) was shown to spread rapidly and produce large syncytium in vitro. HSV-2(SL) and HSV-2(186) were equally susceptible to acyclovir (ACV) and thymine arabinoside (Ara-T). However, HSV-2(SL) was fourfold and 44-fold more susceptible than HSV-2(186) to iododeoxyuridine (IUdR) and bromovinyldeoxyuridine (BVDU), respectively. In addition, cytosolic TK from HSV-2(SL)-infected cells phosphorylated 4, 20, and 23,000 times more IUdR, iododeoxycytidine (IdCyD), and Ara-T than the TK of HSV-2(186), respectively. Further, HSV-2(186) TK did not phosphorylate Ara-T, but HSV-2(186) replication was inhibited by Ara-T. These studies indicate that the retinovirulent HSV-2(SL) has a syn phenotype and a TK with broad substrate activity.

Standardization and demonstration of antibody-coated Candida in urine by direct immunofluorescence test.

Acetone, carbontetrachloride, ethyl alcohol, mixture of ethyl alcohol and acetone, and heat were assessed for fixative property for direct immunofluorescent (IF) staining of antibody-coated Candida cells. The results indicated that ethyl alcohol was the most suitable fixative for the test. Antisera containing 16 units of Candida albicans type A agglutinin were found essential to get optimal detectable fluorescence of antibody-coated yeast cells. IF test showed cross reactivity between the yeasts of C. albicans and C. tropicalis. However, there was no cross reactivity with the conidia of A. flavus. The direct IF test could demonstrate antibody-coated yeast cells and pseudomycelia in deposits of urine in the direct smear. It correlated well with microscopy and culture studies. At times, it could demonstrate the antibody-coated yeasts earlier than routine significant culture. It could also differentiate the significant from non-significant fungal isolates from urine.