Selection of recombinant, library-derived antibody fragments against p24 for human immunodeficiency virus type 1 diagnostics.

By application of combinatorial library technology, we generated the first recombinant antibody fragments directed against the major capsid protein p24 of human immunodeficiency virus type 1 (HIV-1). A library of single-chain Fv fragments (scFvs) was constructed by using the antibody variable-region (V) genes of B cells derived from the spleen of a viral lysate-immunized mouse. Antibodies were selected by panning or by enrichment with biotinylated antigen, yielding four different families of antibody fragments. The different types of scFvs were characterized by affinity measurements, by antigen recognition on Western blots, and by pepscan analysis. The epitope of one of the scFvs is located near the residues involved in CypA binding, thereby making it an attractive candidate for therapeutic applications. Comparison of the V gene sequence of this scFV with that of a previously described monoclonal antibody reactive against this immunodominant epitope revealed the usage of the identical combination of VH and Vkappa regions. Thus, this is one of the rare examples in which the original combination in a library-derived antibody fragment was retrieved. After appropriate affinity and format improvements, the best of our recombinant scFvs may form the basis for a sensitive p24 assay as a measure of viral load. In addition, anti-p24 scFvs could be expressed as intracellular antibodies (intrabodies) to aid in the treatment of HIV infections.

Selection of human anti-human immunodeficiency virus type 1 envelope single-chain antibodies from a peripheral blood cell-based phage repertoire.

Monoclonal antibodies play an important role in the development of diagnostic assays. Instead of using hybridoma technology to isolate human immunodeficiency virus type 1-specific antibodies, a phage-displayed antibody library was generated from a small number (10(7)) of peripheral blood lymphocytes from a seropositive donor. Two families of single-chain antibodies (scFvs) were selected by biopanning with the envelope precursor gp160. ELISA and competition in the BIAcore system revealed that one antibody family recognized a conformation-sensitive epitope within gp120, while the other antibody family was gp41-specific. The latter group had sequence similarity to antibodies recognizing the cluster III epitope of gp41. Binding of scFvs to gp160 could be inhibited with the donor's serum antibodies, indicating that antibodies with a similar specificity were circulating in the donor's blood. Competition experiments suggested that the epitope of the anti-gp41 antibodies was recognized by a broad range of patients' sera: 21 out of 22 sera from North American and all 20 sera from African seropositive patients inhibited binding of scFvs. In contrast, three sera from this panel did not react with the epitope of the anti-gp120 antibodies. These data indicate that, because of the conserved nature of its epitope, the anti-gp41 antibody will be suitable for diagnostic applications.

Post-translational processing of the feline immunodeficiency virus envelope precursor protein.

The synthesis and processing of the envelope glycoprotein precursor of the feline immunodeficiency virus (FIV) isolate FIV-UT113 was investigated in a persistently infected Crandell feline kidney cell line (CRFK) and in an eukaryotic expression system. Pulse-chase studies showed two glycoproteins after a 5 min pulse-labeling: a gp150 and a gp130 species. During a 30-min chase the gp150 species disappeared almost completely while gp130 increased proportionally; it was subsequently processed into the surface glycoprotein (SU), gp100, and the transmembrane glycoprotein (TM), gp35. This final maturation step did also occur when the env gene was expressed independently, but at a much lower rate. The results indicate that FIV-UT113 envelope glycoprotein processing involves two successive proteolytic cleavages. The first cleavage removes a protein fragment of approximately 20 kDa and takes place post-translationally, at least in part. The deduced primary translation product of the env gene lacks an N-terminal signal sequence but instead contains two internal hydrophobic regions. Cleavage is predicted to occur behind the second region which would indeed release an N-terminal 20-kDa polypeptide. Thus, FIV glycoprotein processing resembles that found in the ungulate lentiviruses but differs from that in the primate lentiviruses, the envelope proteins of which possess a short N-terminal signal sequence.

Humoral immune response to feline immunodeficiency virus in cats with experimentally induced and naturally acquired infections.

Sera from cats with naturally acquired and experimentally induced feline immunodeficiency virus (FIV) infections were tested by immunoblot analysis, radioimmunoprecipitation assay (RIPA), and a complex trapping/blocking ELISA. In sequentially obtained samples from experimentally inoculated cats, antibodies against the envelope protein gp120 and the core protein p15 were the first to appear, as indicated by results of RIPA, using lysates of FIV-infected lymphocytes. Antibodies could be detected as early as 2 weeks after infection, followed by a response against p24, p43, and p50. By immunoblot analysis, p24 and p15 were the first proteins detectable between postinoculation weeks 3 and 5; an anti-envelope response was never found by use of this assay, but was found by RIPA. Using the latter test, most sera of naturally infected cats were found to recognize the major core protein p24 in addition to 1 or more minor core proteins. All 40 sera tested precipitated the envelope protein; 3 reacted exclusively with it. A complex trapping/blocking ELISA was developed to quantitate the anti-p24 response. Sera from healthy FIV-infected cats were shown to have higher anti-p24 titer than did those from diseased cats.

Immunogenic peptide comprising a mouse hepatitis virus A59 B-cell epitope and an influenza virus T-cell epitope protects against lethal infection.

The coronavirus spike protein S is responsible for important biological activities including virus neutralization by antibody, cell attachment, and cell fusion. Recently, we have elucidated the amino acid sequence of an S determinant common in murine coronaviruses (W. Luytjes, D. Geerts, W. Posthumus, R. Meloen, and W. Spaan, J. Virol. 63:1408-1412, 1989). A monoclonal antibody directed to this determinant (MAb 5B19.2) protected mice against acute fatal infection. In this study, BALB/c mice were immunized with a synthetic peptide of 13 amino acids corresponding to the binding site of MAb 5B19.2, which was either extended with an amino acid sequence of influenza virus hemagglutinin or conjugated to keyhole limpet hemocyanin. Both immunogens induced S-specific antibodies in mice, but only the hemagglutinin-peptide construct protected them against lethal challenge. In contrast to mouse hepatitis virus type 4 (MHV-4), MHV-A59 was not neutralized in vitro by MAb 5B19.2. Neither MHV-A59 nor MHV-4 was neutralized in vitro by antibodies comprising by the synthetic peptides. Our results demonstrated that antibodies elicited with a synthetic peptide comprising a B-cell epitope and a T-helper cell determinant can protect mice against an acute fetal mouse hepatitis virus infection.

Intracellular proteins of feline immunodeficiency virus and their antigenic relationship with equine infectious anaemia virus proteins.

Feline immunodeficiency virus (FIV) grown in cat lymphocyte and thymocyte cultures was labelled with L-[35S]methionine or [3H]glucosamine and virus-coded proteins were identified using immunoprecipitation. Polypeptides with apparent Mr values of 15K, 24K, 43K, 50K, 120K and 160K were detected. An additional polypeptide of 10K was detected by Western blot analysis. The two highest Mr species sometimes appeared as one band, of which only the 120K polypeptide was glycosylated. In the presence of tunicamycin gp120 was no longer detectable and a non-glycosylated precursor of 75K was found instead. Pulse-chase experiments suggested that the smaller polypeptides p24 and p15 are cleavage products of both p160 and p50. Western blot analysis using a rabbit serum directed against p26 of equine infectious anaemia virus (EIAV) and an anti-EIAV horse serum from a field case of infection revealed a cross-reactivity with p24 of FIV. Cat sera collected late after experimental FIV infection recognized p26 of EIAV, indicating a reciprocal cross-reactivity.

Induction of demyelination by a temperature-sensitive mutant of the coronavirus MHV-A59 is associated with restriction of viral replication in the brain.

The neurovirulence of eight temperature-sensitive (ts) mutants of mouse hepatitis virus strain A59 in 4-week-old BALB/c mice was investigated. Whereas a dose of 100 p.f.u. of wild-type virus killed mice within a week, a 1000-fold higher dose of ts mutants did not. Three ts mutants induced demyelinating disease in the central nervous system (CNS). The pathology of the demyelinating disease caused by one mutant, designated ts-342, was studied in detail. Pathological changes, starting 3 days post-inoculation (p.i.), were characterized by inflammation and demyelination in the CNS. Antibody responses directed against all virus-specific structural proteins were present at 7 days p.i. No virus particles were observed by electron microscopy at 14 days p.i. However, macrophages and lymphocytes were abundant in the areas of demyelination. The growth kinetics in vivo of wild-type virus, ts-342 and a revertant of ts-342 were compared. Wild-type virus and the revertant replicated rapidly in the brain and spread to the liver causing a lethal hepatitis. Ts-342, however, replicated to a much lesser extent within the brain and could not be detected in the blood or liver. The ts lesion in the genome of ts-342 seems, therefore, to determine the outcome of the infection.

Restricted replication of mouse hepatitis virus A59 in primary mouse brain astrocytes correlates with reduced pathogenicity.

Temperature-sensitive (ts) mutants of mouse hepatitis virus A59 (MHV-A59) are drastically attenuated in their pathogenic properties. Intracerebral inoculation of mice with 10(5) PFU of mutant ts342 results in prolonged infection of the central nervous system, whereas 100 PFU of wild-type virus are lethal (M. J. M. Koolen, A. D. M. E. Osterhaus, G. van Steenis, M. C. Horzinek, and B. A. M. van der Zeijst, Virology 125:393-402, 1983). In the Sac(-) cell line ts342 grows as well at 37 degrees C (the body temperature of mice) as at 31 degrees C (the permissive temperature). There is, however, a difference in primary cultures of mouse brain astrocytes. After infection with ts342, astrocytes produced low levels of infectious virus (5.2 +/- 3.7%) compared with virus yields after infection with wild-type virus. The fraction of wild-type virus- and ts342-infected cells was similar. Electron microscopy showed in wild-type virus-infected cells abundant virions in smooth vesicles usually closely associated with a well-developed Golgi apparatus. In mutant-infected cells no mature ts342 virus particles were found. There was no difference between ts342 and wild-type virus regarding the intracellular virus-specific RNAs. In ts342-infected cells the viral glycoproteins E2 and E1 were not detectable or were barely detectable. Either the mRNAs for the glycoproteins are not translated or the proteins are rapidly broken down. Revertants of ts342 were isolated. They grew as well as wild-type virus in astrocytes, indicating that they apparently produced sufficient amounts of E2 and E1, the ts defect itself rather than a second site mutation is responsible for the defect in replication, and the ts defect acts in unison with host-cell factors. The revertants also regained the lethal properties of wild-type virus.

Experimental models of virus attenuation and demyelinating disease.

Several lines of evidence suggest that viruses may be aetiologically related to human demyelinating disorders, but no clear causal relationships have been established. In an effort to understand the pathogenesis of virus-induced CNS disease, animal model systems have been employed. Recent results indicate that mutational changes in viral surface components may profoundly affect the course and outcome of disease.

Virus-induced central positional nystagmus in mice.

Geotropic direction-changing nystagmus in lateral body positions was observed in 4-week-old BALB/c mice after intracerebral injection with a temperature-sensitive mutant of mouse hepatitis virus. The positional nystagmus was detected already 2 days after infection and it lasted half a year at least. The nystagmic responses of the semicircular canals were also evaluated before and after infection. They were unaltered during the disease, which was clinically manifested by general weakness, ataxia and tremor. Histopathological examination 2 weeks after infection revealed demyelination in various parts of the CNS.

Temperature-sensitive mutants of mouse hepatitis virus strain A59: isolation, characterization and neuropathogenic properties.

Twenty 5-fluorouracil-induced temperature-sensitive (ts) mutants of mouse hepatitis virus strain A59 were isolated from 1284 virus clones. Mutants were preselected on the basis of their inability to induce syncytia in infected cells at the restrictive temperature (40 degrees) vs the permissive temperature (31 degrees). Of these mutants, only those with a relative plating efficiency 40 degrees/31 degrees of 3 x 10(-3) or smaller were kept. Virus yields at 40 degrees compared to 37 degrees and 31 degrees (leakiness) were determined. Most mutants (16) were RNA-, i.e., unable to synthesize virus-specific RNA at the restrictive temperature. The other four were RNA+. No qualitative differences were detected in the virus-specific RNAs in cells infected with RNA+ ts-mutants, both at 31 degrees and 40 degrees. Virus-specific proteins present in cells infected with ts-171 (RNA-) and the RNA+-mutants (ts-43, ts-201, ts-209, and ts-279) were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of immunoprecipitates. No qualitative differences in the pattern of virus-specific cellular proteins were detected among the mutants except for an additional polypeptide of about 46,000 daltons in ts-209-infected cells. Finally, the neuropathogenic properties of eight of the mutants were investigated. Whereas 10(2) PFU of wild-type virus injected intracerebrally killed 50 to 100% of 4-week-old Balc/c mice within 1 week, the mutants were highly attenuated. A dose of 10(5) PFU lead to no or transient disease. However, 4 weeks after infection with ts-342, ts-43, or ts-201 obvious histological changes were observed in brain and spinal cord of clinically healthy mice.