Early response gene expression during differentiation of cultured Leishmania donovani.

The promastigote form of the unicellular parasite, Leishmania donovani, must differentiate into the amastigote form to establish an infection in a mammalian host. Identification of genes whose expression changes during differentiation could help reveal mechanisms of Leishmania gene regulation and identify targets for controlling the diseases caused by this human pathogen. Two genomic clones were isolated, P9 that is more highly expressed in promastigotes than in axenic amastigotes and A14 that is preferentially expressed in axenic amastigotes. Analysis of the DNA sequences revealed open reading frames that would encode 55.5 kDa and 100 kDa proteins, respectively, with no homology to known proteins. The mRNA level for these genes during 24 h time courses of parasite differentiation in culture was compared to two genes known to be differentially expressed, c-lpk2 and mkk. Changes in RNA level occurred within 2 h for each gene and continued in advance of morphological changes. The expression levels of these four genes in axenic amastigotes correlated with results from animal-derived parasites.

Genetic analysis of conservation and variation of lipooligosaccharide expression in two L8-immunotype strains of Neisseria meningitidis.

Neisseria meningitidis strains A1 and M978 both express the lipooligosaccharide (LOS) L8 immunotype [Gu et al., J. Clin. Microbiol. 30 (1992) 2047-2053]. Under different growth conditions, strain A1 did not change its LOS profile whereas strain M978 produced variable LOS profiles on SDS-PAGE. To understand the genetic basis of LOS conservation and variation, their lgt locus encoding glycosyltransferases responsible for the biosynthesis of the alpha-chain of LOS was analyzed. Strain A1 possessed only two genes, lgtA and lgtH, at the lgt locus. The lgtA gene was inactivated due to a frameshift mutation; thus strain A1 expressed only L8 LOS. In contrast, strain M978 contained five genes lgtZ, lgtC, lgtA, lgtB and lgtE at this locus, thus it had a potential to express L1, L3,7 in addition to the L8 LOS. The data showed that strain A1 is a better reference strain for the L8 immunotype because of the stability of L8 LOS expression resulting from its unique lgt locus. In addition, these two strains had two new genetic organizations, lgtAH and lgtZCABE, compared to the reported gene organization at the lgt locus in N. meningitidis.

Nucleotide sequences that distinguish Oka vaccine from parental Oka and other varicella-zoster virus isolates.

The sequences of approximately 34 kb from the 3' end of the varicella-zoster virus (VZV) Oka vaccine strain and the previously sequenced Dumas strain were compared. Sequence differences were noted in the coding sequences of several VZV open reading frames (ORFs), including ORFs 48, 51, 52, 55, 56, 58, 59, 60, 62, 64, and 68. Tests based on differences in the ORF62 gene and in the ORF64 poly-A region successfully distinguished the Oka vaccine strain from its wild-type parent and from other Japanese and US clinical isolates. These changes remained stable after passage of the virus in humans.

Generation of HIV-1/HIV-2 cross-reactive peptide antisera by small sequence changes in HIV-1 reverse transcriptase and integrase immunizing peptides.

We have generated peptide antisera against selected regions in HIV-1 and HIV-2 reverse transcriptase (RT) and integrase (IN) to investigate the specificity of determinants governing the immune response. Peptides representing homologous regions (>50%) in the N- and C-termini and central portions of these proteins were synthesized and injected into rabbits. HIV-1 and HIV-2 IN peptide antisera inhibited IN-mediated cleavage of an HIV-1 DNA oligonucleotide substrate in a 3' processing assay, while anti-RT or normal sera had no effect. None of the RT sera inhibited RT activity. In Western blots, HIV-2 antisera directed against RT or IN peptides recognized HIV-2 RT and IN proteins, respectively, as expected, but also cross-reacted with the corresponding HIV-1 proteins. By contrast, corresponding HIV-1 antisera were type-specific. In some cases, HIV-1 cross-reactive antisera could be generated by immunization with HIV-1 chimeric peptides with as few as two residues in the HIV-1 sequence changed to the corresponding HIV-2 amino acids. The finding that a type-specific response can be converted to a cross-reactive response suggests alternate strategies for developing new diagnostic reagents which detect HIV-1 and HIV-2. In addition, our results provide a general model for generating HIV peptide vaccines with dual specificity against HIV-1 and HIV-2.

Susceptibility of the Sf9 insect cell line to infection with adventitious viruses.

Sf9, the insect cell line commonly used for gene expression by recombinant baculovirus (BV), can be infected by St. Louis encephalitis (SLE) virus, a flavivirus, resulting in a persistent, productive, and cytopathic infection, while retaining the ability to be infected with a recombinant baculovirus (rBV). We now demonstrate using double immunofluorescence that single cells are dually infected with SLE virus and rBV. Fourteen additional viruses including additional flaviviruses, other arbovirus classes, vesicular stomatitis virus (VSV), and herpes simplex virus, type 1 (HSV-1) failed to produce a cytopathic effect (CPE) in Sf9 cells. Plaque assays indicated infectious virus was present for several weeks post-inoculation for Yellow fever (YF), Dengue types 1 and 2 (DEN-1 and DEN-2), Gumbo limbo (GL), Eastern equine encephalomyelitis virus (EEE), Western equine encephalomyelitis virus (WEE), HSV-1, and VSV viruses. For HSV-1, GL, EEE, WEE and VSV, but not for YF, DEN-1 or DEN-2 viruses, this could be attributed solely to survival in the Sf9 cell culture media. Of the 14 viruses tested, only HSV-1 could be detected after 2 weeks in serum-free media. The data indicate that several viruses which are pathogenic for humans are stable for long periods of time at 27 degrees C in the serum-containing media used for cultivation of Sf9 cells. YF, DEN-1 and DEN-2 viruses may replicate in Sf9 cells at extremely low levels. This suggests that adventitious agents which do not produce obvious CPE or interfere with rBV infection or recombinant protein expression could contaminate Sf9 cell cultures or media.

Improved immunogenicity of recombinant vaccinia virus-anchored gp120 lacking gp41.

To produce a vaccine against human immunodeficiency virus-1 with improved immunogenicity, the transmembrane and cytoplasmic tail regions of human immunodeficiency virus-1 were replaced with those of the Vesicular Stomatitis Virus glycoprotein, and cloned into vaccinia virus. This recombinant vaccinia virus, vvE13, was compared to one expressing full length envelope gp160, vvE1. Env products of both were located on the cell surface. Antibody response, lymphocyte proliferation and cytotoxicity were better with vvE13 than with vvE1 inoculated mice.

St Louis encephalitis virus establishes a productive, cytopathic and persistent infection of Sf9 cells.

The Sf9 cell line, commonly used for gene expression by recombinant baculovirus, has been productively infected by St Louis encephalitis (SLE) virus, a flavivirus. SLE viral infection produced a c.p.e. in the Sf9 cells characterized by giant cells and the presence of 10-fold fewer cells in the infected cultures after the first week of infection compared with uninoculated control cultures. Infected Sf9 cells expressed SLE viral antigens, and intracellular virus particles were observed by electron microscopy. Titres of cell-associated SLE virus rose slightly over an 8 week period, whereas titres of cell-free virus remained stable, suggesting that SLE virus establishes a productive and persistent infection of Sf9 cells. The SLE virus produced by the Sf9 cells could be neutralized by SLE virus-immune mouse ascitic fluid, and no evidence of escape mutants was detected. Sf9 cells persistently infected with SLE virus could be superinfected with a recombinant baculovirus and expressed recombinant antigen. The successful infection of Sf9 cells by SLE virus represents the first report of production of c.p.e. by SLE virus in insect cells under routine cell culture conditions and of the infection of Sf9 cells by a human pathogen.

Localization of DNA binding activity of HIV-1 integrase to the C-terminal half of the protein.

Human immunodeficiency virus type 1 (HIV-1) integrase (IN) is the viral protein required for integration of the HIV-1 genome into host cell DNA. A series of clones expressing portions of IN as lambda cII fusion proteins has been constructed in an Escherichia coli expression system; a Southwestern procedure was used to examine binding of the expressed proteins to DNA oligonucleotides. Proteins expressed by clone pHIP106, encoding the entire IN protein but no other pol sequence, and pKNA101, which expresses an IN fusion protein containing 23 amino acids of HIV-1 reverse transcriptase at its amino terminus, exhibited similar levels of oligonucleotide binding. Little DNA sequence specificity was associated with binding activity and there was a preference for Mn2+ over Mg2+ and Ca2+. Interestingly, the protein expressed by an N-terminal clone containing nucleotides coding for IN amino acids 1-141 (including a conserved His-Cys box) was unable to bind oligonucleotide, whereas the protein expressed by a C-terminal clone containing nucleotides coding for amino acids 142-288 exhibited binding equivalent to that of full-length IN. The C-terminal protein was unreactive with a MAb to the lambda cII leader peptide and with an antipeptide serum directed against amino acids 141-158. These results are consistent with the previously reported internal initiation of IN protein synthesis in E. coli at met 154, and indicate that the C-terminal clone does not express IN amino acids 142-153. These amino acids represent part of a conserved region termed D(35)E, containing amino acids 116-152, which has been implicated in IN DNA binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Mapping of the epitopes of Epstein-Barr virus gp350 using monoclonal antibodies and recombinant proteins expressed in Escherichia coli defines three antigenic determinants.

The Epstein-Barr virus (EBV) major surface membrane antigen (MA), gp350/220, induces antibodies that neutralize virus infectivity in vitro. The MA glycoprotein is encoded by nucleotides 1784 to 4504 of the BamHI L fragment of the EBV genome. To define the antigenic epitopes on gp350, sequences encoding portions of the protein were cloned into an Escherichia coli expression system and eight recombinant clones were generated, two overlapping clones representing the C terminus and six overlapping clones representing the N terminus. The epitopes expressed by the recombinant proteins were mapped using 14 anti-MA monoclonal antibodies (MAbs) in a dot blot immunoassay. One of the MAbs reacted with clones that express the C terminus of gp350 and three others reacted with clones expressing the N terminal portion of the protein; the remaining MAbs tested were not reactive with the cloned proteins. The data identify three antigenic determinants on gp350. DNA sequences encoding these epitopes are located between nucleotides 1980 and 2307, 3186 and 3528, and 3528 and 3576 of the BamHI L fragment. In an attempt to elicit neutralizing antibodies, rabbits were immunized with gel-purified recombinant proteins from four of the clones. Neutralization assays indicate that the proteins expressed by these clones do not induce in vitro virus-neutralizing antibodies.

Expression of HIV-1 integrase in E. coli: immunological analysis of the recombinant protein.

Sequences encoding the human immunodeficiency virus type 1 (HIV-1) integrase gene have been cloned and expressed in Escherichia coli. The expressed protein is a lambda cII fusion protein of 37 kD containing the carboxyl-terminal 23 [corrected] amino acids of reverse transcriptase fused to the entire integrase sequence and is insoluble, a feature which allows partial purification away from soluble bacterial proteins. As judged by its reactivity with HIV positive sera in Western blot and in enzyme-linked immunosorbent assay (ELISA), the recombinant integrase retains antigenicity similar to native protein. Additionally, ELISA data obtained with the cloned protein indicate that patients infected with HIV-1 who are at different stages of progression to AIDS have antibodies reactive with the cloned integrase. HIV-2 positive human sera are also reactive with the cloned integrase. Rabbit antibodies produced against the recombinant protein react both by ELISA and Western blot with the homologous bacterially expressed protein, recognize both virion HIV-1 integrase and reverse transcriptase in Western blots, and immunoprecipitate an HIV-1 virion protein of 34 kD. Unlike human antisera from patients infected with HIV-1 or HIV-2 which are frequently reactive with both HIV-1 and HIV-2 integrase, the rabbit antibodies are type specific, reacting with HIV-1, but not with HIV-2 integrase by Western blot.

Epitope mapping of the HIV-1 gag region by analysis of gag gene deletion fragments expressed in Escherichia coli defines eight antigenic determinants.

Immune response to HIV infection is generally characterized by appearance of antibodies to the gag protein p24 early in infection, and by apparent loss of p24 antibodies accompanied by increases in p24 antigen levels with disease progression. Precise definition of the immunodominant epitopes present in gag gene proteins has potential clinical significance. Seventeen anti-gag monoclonal antibodies (MAb) were used in enzyme-linked immunosorbent assays (ELISA) with antigens expressed by nine recombinant clones to define epitopes on HIV gag proteins which elicit an immune response. All of the MAbs tested, except two anti-p17, reacted with a clone which expresses the carboxyl terminal 13 amino acids of p17 and all of p24 and p15. All anti-p24 MAbs reacted with clones containing all of p24. MAbs reacted differentially with clones containing deleted regions depending on the antigenic portion expressed. Of thirteen potential identifiably different genomic regions which could be predicted from the genomic structure of the clones, eight different antigen epitopes were defined: two on p17, five on p24, and one on p15 (in the region corresponding to the carboxyl terminal protein p6). Six regions did not appear to react with any of the monoclonal antibodies available. Identification of the epitopes present in the cloned antigens should allow their use to evaluate sera from HIV-infected donors at different clinical stages of progression to AIDS.

Quantitation of poliovirus antigens in inactivated viral vaccines by enzyme-linked immunosorbent assay using animal sera and monoclonal antibodies.

Recent advances in methods for the manufacture of inactivated poliovirus vaccines have resulted in increased vaccine immunogenicity. In conjunction with this capability it is important to have available highly sensitive and quantitative potency assays. The potential suitability of enzyme-linked immunoassay (ELISA) was evaluated using animal sera with neutralizing antibodies or neutralizing monoclonal antibodies for antigen detection in potency tests. The monoclonal antibodies developed, which bound D antigen but not C antigen, were neutralizing unless relatively weakly reactive. Those that bound C antigen only were non-neutralizing. Those that bound both C and D antigens were sometimes neutralizing. D-specific and D/C-specific neutralizing monoclonal antibodies against type-2 poliovirus protected mice on passive immunization against paralytic disease and death from the MEF strain virus. Potency measurements by ELISA using either D-specific neutralizing monoclonal antibodies or type-specific goat sera for antigen detection were sensitive and precise. Tests using C-specific monoclonal antibodies for antigen detection indicated that increased C antigen content may result in falsely elevated reactivities of animal sera with some vaccines. Monoclonal antibodies may be useful ELISA reagents for IPV potency testing.

Reactivity of an HIV gag gene polypeptide expressed in E. coli with sera from AIDS patients and monoclonal antibodies to gag.

A segment of the gag gene of the human immunodeficiency virus (HIV) (HTLV-IIIB strain), the virus which causes acquired immunodeficiency syndrome (AIDS), has been cloned into the bacterial expression vector, pCQV2, and mapped to the right-hand portion of the gag gene containing the carboxyl-terminal portion of p24 and the amino-terminal portion of p15. Nucleic-acid sequencing of the insert-vector junctions further defined the 5'-terminal nucleotide of HIV sequence as nucleotide 997 and the 3'-terminal nucleotide as 1696. When used in an enzyme-linked immunosorbent assay (ELISA) with sera from HIV-infected patients, the cloned antigen reacted with a subset of sera which were positive on a standard ELISA using whole virus as antigen. Western-blot screening of these sera with whole virus indicated that all p24-positive sera were positive with the clone, suggesting that the carboxyl-terminal portion of p24 contains a highly antigenic epitope(s). A serum which was p24-negative p15-positive by Western blot analysis was also highly reactive, indicating that a p15 epitope is present in the cloned antigen. Epitope mapping with a series of monoclonal antibodies to gag resulted in positive ELISA with 2 of 3 anti-p24, 0 of 1 anti-p15, and 0 of 1 anti-p17 Western-blot-positive monoclonal antibodies, suggesting that one of the anti-p24 monoclonal antibodies reacts with epitopes amino-terminal to those coded from nucleotide 997, two anti-p24 monoclonals react with epitopes carboxyl-terminal to those coded from nucleotide 997, and the anti-p15 monoclonal reacts with epitopes carboxyl-terminal to those coded from nucleotide 1696.

Poliovirus and polio antibody assay in HEp-2 and Vero cell cultures.

HEp-2 cell cultures were about three to 30 times more sensitive for poliovirus titration than Vero cells. Attenuated strains induced a complete cytopathic effect in HEp-2 but not in Vero cells. For polio antibody titration, HEp-2 and Vero cells were equally suitable. A high degree of sensitivity and reproducibility of virus neutralization was achieved in tests utilizing a low virus dose and serum-virus incubation overnight at 36 degrees C. Staining of infected trays with crystal violet obviated reading of viral CPE under the microscope and expedited the evaluation of larger-scale tests.

Encephalitogenicity of measles virus in marmosets.

Marmosets infected intracerebrally with the wild Edmonston strain of measles virus developed encephalitis, demonstrated histologically and by the fluorescent-antibody technique. The infection remained clinically silent over a 14-day observation period. Animals infected intracerebrally with the JM strain of wild measles virus had only mild encephalitic changes but died of the visceral form of measles infection. Marmosets inoculated with measles vaccine had no encephalitis and remained clinically well. Marmosets appear to be a sensitive indicator of the viscerotropic and neurotropic properties of measles virus.

Sensitive hemagglutination inhibition test for mumps antibody.

Incorporation of heterologous immunoglobulin against human immunoglobulin G into the mumps HI tests provided a highly sensitive and specific way of measuring mumps antibody in human sera. The sensitivity of this enhanced mumps HI test compared favorably with that of an anti-immunoglobulin-enhanced virus plaque neutralization test, but the HI test required considerably less time and effort to perform.

Fatal measles infection in marmosets pathogenesis and prophylaxis.

Moustached marmosets (Saguinus mystax) were infected intranasally with either of two low-passaged, wildlike strains of measles virus, strain Edmonston or strain JM. The infection resulted in 25 and 100% mortality, respectively, 12 to 14 days after infection. Clinical signs, gross pathological findings, and histology lacked the characteristic features of measles in other primates. A deficient immune response and widespread gastroenterocolitis appeared to be the main causes for the fatal outcome. Fluorescent-antibody staining detected large amounts of measles antigen in lymphatic tissues, the gastrointestinal and respiratory tracts, the salivary glands, pancreas, liver, kidney, and other visceral tissues. Live attenuated or inactivated measles vaccine proved equally effective in preventing fatal measles in marmosets. Challenge with live virus of animals which were primed 1 year previously with inactivated alum-absorbed vaccine resulted in a precipitous response, with a 100- to 1,000-fold increase in antibody titers. This vigorous booster response suggests the existence of a primary deficiency in lymphocyte cooperation in marmosets, which upon adequate priming is followed by extensive clonal expansion and antibody synthesis. Marmosets appear to be the most susceptible primate species to measles infection. They are capable of distinguishing differences in virulence of virus strains with a level of sensitivity not available in other animals.