Sequence analysis of the ank gene of granulocytic ehrlichiae.

The ank gene of the agent of human granulocytic ehrlichiosis (HGE) codes for a protein with a predicted molecular size of 131.2 kDa that is recognized by serum from both dogs and humans infected with granulocytic ehrlichiae. As part of an effort to assess the phylogenetic relatedness of granulocytic ehrlichiae from different geographic regions and in different host species, the ank gene was PCR amplified and sequenced from a variety of sources. These included 10 blood specimens from patients with confirmed human granulocytic ehrlichiosis (three from New York, four from Wisconsin, two from Slovenia, and one from Sweden). Also examined was a canine granulocytic ehrlichia sample obtained from Minnesota, Ehrlichia equi from California, Ehrlichia phagocytophila from Sweden, and the granulocytic ehrlichia isolate USG3. The sequences showed a high level of homology (>95.5% identity), with the lowest homology occurring between a New York HGE agent and the Swedish E. phagocytophila. Several 3-bp deletions and a variable number of 51- and 81-bp direct repeats were noted. Although the North American HGE sequences showed the highest conservation (>98.1% identity), phylogenetic analyses indicated that these samples represent two separate clades, one including the three New York HGE samples and the USG3 strain and another with the Wisconsin HGE and Minnesota canine sequences. Two of the New York samples and the USG3 strain showed 100% identity over the entire 3,696-bp product. Likewise, three of the Wisconsin human samples and the Minnesota dog sample were identical (3,693 bp). Whereas phylogenetic analysis showed that the E. equi sequence was most closely related to the Upper Midwest samples, analysis of the repeat structures showed it to be more similar to the European samples. Overall, the genetic analysis based on the ank gene showed that the granulocytic ehrlichiae are closely related, appear to infect multiple species, and can be grouped into at least three different clades, two North American and one European.

Major antigenic proteins of the agent of human granulocytic ehrlichiosis are encoded by members of a multigene family.

Western blot analysis of proteins from a cell culture isolate (USG3) of the human granulocytic ehrlichiosis (HGE) agent has identified a number of immunoreactive proteins, including major antigenic proteins of 43 and 45 kDa. Peptides derived from the 43- and 45-kDa proteins were sequenced, and degenerate PCR primers based on these sequences were used to amplify DNA from USG3. Sequencing of a 550-bp PCR product revealed that it encodes a protein homologous to the MSP-2 proteins of Anaplasma marginale. Concurrently, an expression library made from USG3 genomic DNA was screened with granulocytic Ehrlichia (GE)-positive immune sera. Analysis of two clones showed that they contain one partial and three full-length highly related genes, suggesting that they are part of a multigene family. Amino acid alignment showed conserved amino- and carboxy-terminal regions which flank a variable region. The conserved regions of these proteins are also homologous to the MSP-2 proteins of A. marginale; thus, they were designated GE MSP-2A (45 kDa), MSP-2B (34 kDa), and MSP-2C (38 kDa). The PCR fragment obtained as a result of peptide sequencing was completely contained within the msp-2A clone, and all of the sequenced peptides were found in the GE MSP-2 proteins. Recombinant MSP-2B protein and an MSP-2A fusion protein were expressed in Escherichia coli and reacted with human sera positive for the HGE agent by immunofluorescence assay. These data suggest that the 43- and 45-kDa proteins of the HGE agent are encoded by members of the GE MSP-2 multigene family.

Molecular cloning and sequencing of three granulocytic Ehrlichia genes encoding high-molecular-weight immunoreactive proteins.

Granulocytic Ehrlichia was isolated from canine blood obtained from animals challenged with field-collected Ixodes scapularis and propagated in HL60 cells. PCR primers specific for the 16S ribosomal DNA (rDNA) of the Ehrlichia genogroup comprising E. equi, E. phagocytophila, and the agent of human granulocytic ehrlichiosis (HGE) amplified DNA from extracts of these cells. Sequence analysis of this amplified DNA revealed that it is identical to the 16S rDNA sequence of the HGE agent. A genomic library was constructed with DNA from granulocytic Ehrlichia and screened with pooled sera from tick-challenged, granulocytic Ehrlichia-infected dogs. Several clones were isolated and sequenced. Three complete genes encoding proteins with apparent molecular masses of 100, 130, and 160 kDa were found. The recombinant proteins reacted with convalescent-phase sera from dogs and human patients recovering from HGE. This approach will be useful for identifying candidate diagnostic and vaccine antigens for granulocytic ehrlichiosis and aid in the classification of genogroup members.

Safety, efficacy, and immunogenicity of a recombinant Osp subunit canine Lyme disease vaccine.

A subunit canine Lyme disease vaccine formulated with recombinant lipidated Osp A and OspB and saponin QS21 was assessed for safety, protective efficacy, and immunogenicity. Ten normal beagles were subcutaneously vaccinated twice at age 12 and 16 weeks, respectively. Three months after the second vaccination, the vaccinates and another 10 nonvaccinated control beagles were challenged by feeding ticks on each dog for 5 days using eight field-collected adult female and six adult male Ixodes scapularis infected with Lyme disease spirochetes per dog. Adverse reactions associated with the vaccinations were limited to injection site swellings which occurred within the first 48 h and resolved within a week. The local reaction was independent of vaccination times and tick challenge. On the basis of typical clinical signs, xenodiagnosis, and diagnostic immunoblotting, all 10 controls were infected; five developed lameness and three of them experienced at least two to three episodes of limping during a 10-month monitoring period. In contrast, eight of ten vaccinates were protected and two infected vaccinates, as judged by xenodiagnosis, were asymptomatic. None of the protected vaccinates developed antibodies to diagnostic spirochetal antigens other than OspA and OspB. In contrast, most controls produced antibodies to borrelial antigens, but not to OspA and OspB. Antibody production in vaccinates receiving a third vaccination 10 months postchallenge was greatly boosted; the geometric mean antibody titer was significantly higher (P < 0.0001) than that tested prechallenge. Thus, the subunit canine Lyme disease vaccine was safe and protective and elicited immunological memory. Vaccinated dogs were serologically distinguishable from those naturally exposed.

Saponin adjuvant enhancement of antigen-specific immune responses to an experimental HIV-1 vaccine.

The adjuvant activity of a single highly purified saponin from the soap bark tree Quillaja saponaria was evaluated by using it as a component in an experimental vaccine containing rHIV-1 envelope protein (HIV-1 160D) adsorbed to alum. BALB/c mice immunized with experimental vaccine formulations containing the saponin adjuvant QS-21 produced significantly higher titers of antibodies than mice vaccinated with only the alum-adsorbed HIV-1 160D. Potent amnestic antibody responses to HIV-1 viral proteins were also induced. Ag-specific proliferative responses to recombinant proteins and to three variants of HIV-1 were significantly increased using QS-21 as an adjuvant. Alum-adsorbed HIV-1 160D failed to induce measurable proliferative responses to inactivated HIV-1 viruses, but group-specific proliferative responses were raised when the QS-21 adjuvant was used in the vaccine formulation. MHC class I restricted CTL specific for the immunodominant V-3 loop were induced but only when the QS-21 adjuvant was included in the vaccine formulation. The production of serine esterase by Ag-activated splenic mononuclear cells, indicating the maturation of precursor CTL, was used as a secondary measure of CTL activity, and this response was also increased. The specificity of antibody responses was not significantly broadened using QS-21; the adjuvant increased the immune recognition of epitopes throughout the HIV-1 glycoprotein 160. However, the specificity of the proliferation and serine esterase responses was broadened, suggesting that the QS-21 augmented cell-mediated immune responses specific for epitopes outside of the V-3 loop. Additionally, the QS-21 adjuvant appeared to induce recognition of weakly immunogenic epitopes that were not recognized using only alum-adsorbed HIV-1 160D. The ability of QS-21 to augment both antibody and cell-mediated immune responses suggests that this adjuvant could be a valuable component in subunit vaccines.

Development of a genetically engineered vaccine against feline leukemia virus infection.

A genetically engineered subunit vaccine against FeLV infection was developed. The protective immunogen in the vaccine was a purified recombinant protein containing the entire amino acid sequence of FeLV subgroup A gp70 envelope protein. The optimal adjuvant was determined to be a highly purified saponin, QS-21, derived from Quillaja saponaria Molina. A vaccine formulation containing the recombinant protein, QS-21, and aluminum hydroxide was tested in specific-pathogen-free kittens and was shown to induce neutralizing antibodies as well as appreciable antibody responses to native gp70 by enzyme immunoassay and protein (western) immunoblot analysis and of whole virus preparations.

Genetically-engineered subunit vaccine against feline leukaemia virus: protective immune response in cats.

A recombinant retroviral subunit vaccine has been developed that successfully protects cats from infectious feline leukaemia virus (FeLV) challenge. The antigen used is a non-glycosylated protein derived from the envelope glycoprotein of FeLV subgroup A, expressed in Escherichia coli. This recombinant protein, rgp70D, includes the entire exterior envelope protein gp70, plus the first 34 amino acids from the transmembrane protein p15E. The vaccine consists of purified rgp70D absorbed on to aluminium hydroxide and used in conjunction with a novel saponin adjuvant. Cats immunized with this formulation developed a strong humoral immune response, including neutralizing and feline oncornavirus-associated cell membrane antigen antibodies. Vaccinated animals showed an anamnestic response upon intraperitoneal challenge with FeLV-A, and were protected from viral infection. In contrast, the control animals developed viraemia shortly after the challenge, which in most cases became chronic. Formulation of the same antigen with other widely used adjuvants elicited poor protective immune responses in cats.

Temporal expression of HIV-1 envelope proteins in baculovirus-infected insect cells: implications for glycosylation and CD4 binding.

Three different human immunodeficiency virus type I (HIV-1) envelope derived recombinant proteins and the full length human CD4 polypeptide were expressed in Spodoptera frugiperda (Sf9) cells. DNA constructs encoding CD4, gp120, gp160, and gp160 delta (full length gp160 minus the transmembrane and cytoplasmic region of gp41) were cloned into the baculovirus expression vector pVL941 or a derivative and used to generate recombinant viruses in a cotransfection with DNA from Autographa californica nuclear polyhedrosis virus (AcMNPV). Western blotting of cell extracts of the recombinant HIV-1 proteins showed that for each construct two major bands specifically reacted with anti-HIV-1 envelope antiserum. These bands corresponded to glycosylated and nonglycosylated versions of the HIV proteins as determined by 3H-mannose labeling and tunicamycin treatment of infected cells. A time course of HIV envelope expression revealed that at early times post-infection (24 hours) the proteins were fully glycosylated and soluble in nonionic detergents. However, at later times postinfection (48 hours), expression levels of recombinant protein reached a maximum but most of the increase was due to a rise in the level of the nonglycosylated species, which was largely insoluble in nonionic detergents. Thus, it appears that Sf9 cells cannot process large amounts of glycosylated recombinant proteins efficiently. As a measure of biological activity, the CD4 binding ability of both glycosylated and nonglycosylated recombinant HIV envelope proteins was tested in a coimmunoprecipitation assay. The results showed that CD4 and the glycosylated versions of recombinant gp120 or gp160 delta specifically associated with one another in this analysis. Nonglycosylated gp120 or gp160 delta proteins from tunicamycin-treated cultures did immunoprecipitate with anti-HIV-1 antiserum but did not interact with CD4. We conclude that production of native HIV envelope proteins, as measured by addition of carbohydrate side chains and ability to bind CD4, peaks early after infection in baculovirus-infected insect cells.

Strong sequence conservation among horizontally transmissible, minimally pathogenic feline leukemia viruses.

We report the first complete nucleotide sequence (8,440 base pairs) of a biologically active feline leukemia virus (FeLV), designated FeLV-61E (or F6A), and the molecular cloning, biological activity, and env-long terminal repeat (LTR) sequence of another FeLV isolate, FeLV-3281 (or F3A). F6A corresponds to the non-disease-specific common-form component of the immunodeficiency disease-inducing strain of FeLV, FeLV-FAIDS, and was isolated from tissue DNA of a cat following experimental transmission of naturally occurring feline acquired immunodeficiency syndrome. F3A clones were derived from a subgroup-A-virus-producing feline tumor cell line. Both are unusual relative to other molecularly cloned FeLVs studied to date in their ability to induce viremia in weanling (8-week-old) cats and in their failure to induce acute disease. The F6A provirus is organized into 5'-LTR-gag-pol-env-LTR-3' regions; the gag and pol open reading frames are separated by an amber codon, and env is in a different reading frame. The deduced extracellular glycoproteins of F6A, F3A, and the Glasgow-1 subgroup A isolate of FeLV (M. Stewart, M. Warnock, A. Wheeler, N. Wilkie, J. Mullins, D. Onions, and J. Neil, J. Virol. 58:825-834, 1986) are 98% homologous, despite having been isolated from naturally infected cats 6 to 13 years apart and from widely different geographic locations. As a group, their envelope gene sequences differ markedly from those of the disease-associated subgroup B and acutely pathogenic subgroup C viruses. Thus, F6A and F3A correspond to members of a highly conserved family and represent prototypes of the horizontally transmitted, minimally pathogenic FeLV present in all naturally occurring infections.

Detection of antibodies to human immunodeficiency virus by latex agglutination with recombinant antigen.

Recombinant human immunodeficiency virus (HIV) env antigen was attached to polystyrene particles, and these complexes were used to develop the first latex agglutination assay for antibodies to HIV. A total of 95 positive and 116 negative human serum samples were assayed for antibodies to HIV by latex agglutination, and results were compared with those of a commercial enzyme immunoassay. Latex agglutination was also compared with, and found to be completely concordant with, Western blot (immunoblot) analysis with virion antigens.

Enzyme immunoassay using a novel recombinant polypeptide to detect human immunodeficiency virus env antibody.

A unique antigen, CBre3, has been synthesized from a genetically engineered clone to detect human immunodeficiency virus (HIV) env antibodies with high sensitivity and specificity. The antigen contains sequences derived from both envelope proteins of HIV, i.e., gp120 and gp41, and was purified free of Escherichia coli proteins detectable by Coomassie stain or immunoblotting with E. coli antiserum. The purified recombinant polypeptides were used as antigen in an enzyme immunoassay (EIA) to screen serum samples from healthy and HIV-infected individuals. The same samples were also tested by radioimmunoprecipitation (RIP) for gp120 and gp160 HIV antibodies. All samples containing gp120 and gp160 antibodies by RIP had CBre3 EIA values greater than 0.35 (n, 122; range, 0.37 to 2.1+; median, 1.65). All RIP HIV antibody-negative samples had CBre3 EIA values less than 0.25 (n, 140; mean, 0.052; standard deviation, 0.045; range, 0.00 to 0.22). The endpoint titer of a standard positive control serum was 1:10,000 by RIP and by CBre3 EIA. The assay was 100% accurate in three proficiency panels. It easily detected six samples from individuals whose infections were confirmed by culture; these samples were reactive only with p24 by Western blot. The samples also were positive for gp120 and gp160 antibodies by RIP. These data suggest that the CBre3 EIA can detect env antibodies as sensitively and specifically as RIP and with more sensitivity than Western blot.

Diagnosis of human immunodeficiency virus infection by immunoassay using a molecularly cloned and expressed virus envelope polypeptide. Comparison to Western blot on 2707 consecutive serum samples.

To detect human immunodeficiency virus (HIV) antibodies in a simple enzyme-linked immunoassay (CBre3-EIA), we used an Escherichia coli-expressed polypeptide antigen, representing the carboxy-terminal third of the external membrane glycoprotein gene fused with the amino-terminal half of the transmembrane glycoprotein gene. Over a 3-month period, 2707 consecutive serum samples referred for confirmatory testing for human T-lymphotrophic virus type III (HTLV-III) antibodies were evaluated by both Western blot and CBre3-EIA. On a single determination for each sample, the CBre3-EIA was found to have an estimated sensitivity (99.9%) and specificity (99.1%) similar or superior to the more cumbersome Western blot method. This study shows that all HIV-seropositive subjects have antibodies to the virus envelope protein; no other virus antigens are required for construction of highly sensitive immunoassays.

Human retroviral env and gag polypeptides: serologic assays to measure infection.

Humoral antiviral responses to human retrovirus infections identify persistently infected individuals and can be used to characterize virus-host interactions. Antibodies to native viral polypeptides have been reliably measured, although quantitation of env antibodies is difficult due to a lack of purified antigens. To quantitate antibodies to env antigens, bacterially expressed cloned env polypeptides from the transmembrane regions of human T lymphotropic virus types I and III were applied to nitrocellulose filters in an immunodot assay. A combination of the sensitivity of the Western blot procedure and the specificity of peptides from defined viral sequences was used to detect 49/49 HTLV-III/LAV-infected individuals previously defined as seropositive by radioimmunoprecipitation sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Of these HTLV-III/LAV envelope seropositive people, 22% lacked antibody to p24 in a radioimmunoassay. In contrast, the sensitivity of antibody detection to HTLV-I env antigens and p24 were comparable. Antibodies to HTLV-I and HTLV-III/LAV env transmembrane peptides were not cross-reactive. Levels of antibody to env antigens of both HTLV-I and HTLV-III/LAV persisted without change for at least 26 mo, suggesting that most infections represent stable virus-host interactions. The use of bacterially expressed env peptides offers a rapid serologic approach for distinguishing human retroviral infections and can be used to define immune responses to specific regions of the viral genome.

The expression of the SV40 early region in the transformed human cell line SV80.

The one complete copy of the SV40 early region present in human cells of the transformed line SV80 carries a duplication of the 5' portion of the early region, including its transcriptional control region and splicing signals (W. Gish and M. Botchan, personal communication). Novel SV40-specific RNA species of sufficiently large size, 3.8 and 4.2 kb, to be expressed from the duplicated early transcriptional control region were detected in SV80 cytoplasmic and nuclear RNA preparations by blot hybridization. The results of transcription in a cell-free system of a plasmid, pSV80-04, representing this SV80 cell SV40 DNA integrate (W. Gish and M. Botchan, personal communication) and of nuclease protection experiments with end-labelled pSV80-04 DNA fragments support the conclusion that the duplicated early sequences are transcribed in SV80 cells. It has also been established that the duplicated early splicing signals are functional in SV80 cells. These results are discussed in relation to the large amounts of SV40 early mRNA and T-antigen synthesized in cells of the SV80 line.

Synthesis and processing of simian virus 40-specific RNA in adenovirus-infected, simian virus 40-transformed human cells.

Human simian virus 80 (SV80) cells transformed by simian virus 40 (SV40) synthesize substantial quantities of the SV40 large T-antigen (Henderson & Livingston, 1974; Tjian, 1978) and cytoplasmic, poly(A)-containing RNA species that exhibit spliced structures characteristic of the SV40, early messenger RNA species that encode both large and small T-antigens (Flint & Beltz, 1979). When SV80 cells were infected with type C adenovirus, both the synthesis of SV40 large T-antigen and the appearance in the cytoplasm of newly synthesized, SV40-specific RNA sequences were inhibited during the late phase of infection. The results of hybridization to SV40 DNA of SV80 nuclear RNA, prepared from mock- or adenovirus-infected cells after labeling for short periods in vivo or in vitro, indicated that transcription of integrated SV40 was, by contrast, not disrupted during the late phase of adenovirus infection. Poly(A)-containing, nuclear RNA species that hybridized to SV40 DNA sequences and exhibited the sizes of spliced, large and small T-antigen mRNA species were also synthesized in infected cells at a time when the corresponding mRNA sequences did not leave the nucleus. These results suggest that the failure of non-adenoviral mRNA sequences to enter the cytoplasm of adenovirus-infected cells does not reflect inhibition of either their transcription or the normal enzymatic processing reactions to which pre-mRNA species are subject. Several lines of evidence do, however, establish that nuclear, SV40-specific RNA sequences are less stable in adenovirus-infected compared to mock-infected SV80 cells.