Characterization of monoclonal antibodies specific for sequential influenza A/PR/8/34 virus variants

A panel of monoclonal antibodies specific for a corresponding panel of sequentially selected variants of influenza A/PR/8/34 virus has been established. Although the monoclonal antibodies are paratypically distinct, idiotypic relatedness has been observed. Two cross-reactive idiotypes have been defined that are associated with the 7183 and S107 VH gene families, respectively. Three of the four monoclonal antibodies utilize the VK21 group of light chains, and three VH genes belong to the VH7183 family and one to the VH S107 family. Antibodies encoded by genes deriving from the VH7183 family share a cross-reactive idiotype, a marker of the VH region as well as distinct individual idiotopes. These antibodies are produced by different clones using related VH and VK genes.     

Many variable region genes are utilized in the antibody response of BALB/c mice to the influenza virus A/PR/8/34 hemagglutinin

We have examined how many different H chain variable (VH) and kappa-chain variable (Vk) germ-line genes are used in the antibody response to the influenza virus A/PR/8/34 hemagglutinin (PR8 HA), and have assessed how the expression of individual VH and/or Vk genes contributes to the generation of specificity for the HA. A panel of 51 hybridoma antibodies that recognize two antigenic regions on the HA were compared for the sequence of their Ig H and L chain V regions. The hybridomas were obtained from 28 individual BALB/c mice that had been immunized with PR8 under a variety of primary and secondary response immunization protocols. The degree and pattern of sequence similarity suggests that 29 different VH genes drawn from seven different VH gene families, and 25 different Vk genes drawn from 12 different Vk gene families were used in this panel. Based on current estimates of the total numbers of VH and Vk genes in the mouse, this suggests that between 2.5 and 10% of the entire VH and Vk germ-line repertoires were used by these hybridomas. Despite this extensive diversity, some V genes were repetitively identified among these hybridomas, and were most often expressed in the context of specific VH/Vk combinations. Because antibodies that used identical VH/Vk combinations also usually displayed similar reactivity patterns with a panel of mutant viruses, this indicates that VH/Vk pairing can be important in establishing the specificity of antibodies for the HA.     

Sequences of the VH and VL regions of murine monoclonal antibodies against 3-fucosyllactosamine

Many mAb that bind the carbohydrate antigenic determinant 3-fucosyl-lactosamine (3-FL), Gal beta 1-4[Fuc alpha-3]GlcNAc-R have been raised in BALB/c mice, and we are studying the structure and regulation of these antibodies. In this report, we present the first information about their amino acid sequences and the Ig gene segments used to encode them. V regions of the H and L chains of three anti-3-FL antibodies, PMN6, PMN29, and PM81, were sequenced by a combination of mRNA and amino acid sequencing. The L chain sequences of PMN6 and PM81 antibodies indicate that their VK and JK regions are encoded by VK24B and JK1 germ-line genes, respectively. The nucleotide and amino acid sequences of the H chains suggest that the three anti-3-FL antibodies are encoded by the VH441 gene segment of the X24 VH family, and this conclusion was supported by Southern filter hybridization with VH441 and JH3-JH4 probes. PMN29 has at least 11 amino acid substitutions, which is an unusually large amount of somatic mutation for an IgM antibody. Previous analyses of BALB/c genomic libraries with VHX24 and VH441 probes make it unlikely that this VH family contains additional germ-line genes, but this possibility cannot be excluded. All three antibodies use the DQ52 and JH4 gene segments. The single VH and VL gene segments used to encode the anti-3-FL antibodies is in contrast to the multiple VH and VL segments used by antibodies against other carbohydrate Ag such as alpha 1-6 dextran and group A streptococcal carbohydrate. VH441 also encodes the VH regions of antibodies against galactan and levan (beta 2-6 fructosan). The similarities among VH segments of antibodies against 3-FL, levan, and galactan, and the striking differences in their CDR3 sequences, suggest that CDR3 plays an important role in the formation of the Ag binding site. The use of a single VH segment from the smallest VH gene family by antibodies against at least three different carbohydrate determinants is noteworthy. It raises the possibility that the amino acid sequence encoded by VH441 has some general structural features that make it particularly well adapted for binding to carbohydrate sequences.     

Complete nucleotide sequence of the influenza A/PR/8/34 virus NS gene and comparison with the NS genes of the A/Udorn/72 and A/FPV/Rostock/34 strains.

The nucleotide sequence of the NS gene of the human influenza virus A/PR/8/34 was determined and found to be the same length (890 nucleotides) as the NS gene of another human influenza virus A/Udorn/72 and of the avian isolate A/FPV/Rostock/34. Comparison of the sequences of the NS genes of the two human influenza viruses shows an 8.9% difference whereas the NS gene of the avian isolate differs by only 8% from that of the human strain A/PR/8/34. The extensive sequence similarity among these three genes does not support the notion of species specific homology groups among NS genes of avian and human influenza virus strains. The primary sequence of the A/PR/8/34 NS gene is consistent with the findings that the influenza virus NS gene may code for two overlapping polypeptides. In addition, an open reading frame potentially coding for a polypeptide 167 amino acids in length was found in the negative strand RNA of the A/PR/8/34 virus NS gene.     

A rabbit antiserum detects a VH J558 subgroup marker highly expressed among anti-alprenolol antibodies

A rabbit antiserum raised against anti-alprenolol mAb 14C3 detects common antigenic determinants (ADC3) in 10 out of 14 anti-alprenolol mAb that use different germ-line VH and/or Vk genes. The anti-14C3 antiserum binds only to H chains in immunoblots, therefore suggesting that at least part of the ADC3 determinants may be encoded by H chain V region genes. Analysis of VH gene family usage among the anti-alprenolol mAb reveals that the expression of ADC3 correlates with utilization of VH genes that belong to the J558 gene family, regardless of the JH, Vk, and Jk genes. To determine whether the ADC3 determinants are general V region markers or whether they are unique to anti-alprenolol antibodies, we have extended our analysis to a random panel of antibodies that also use VH genes of the J558 family. Among 23 mAb of various specificities, 14 react with the anti-14C3 antiserum in immunoblot and in ELISA, irrespective of antibody specificity. Adsorption of the antiserum on one of these positive antibodies results in a loss of reactivity toward both anti-alprenolol and unrelated antibodies. Therefore, several but not all antibodies that use a J558 VH gene also express the complete set of epitopes defining ADC3. These results strongly suggest that ADC3 are markers of a subset of J558 VH gene products. The anti-14C3 antiserum may thus constitute a "serologic probe" for identification of a VH gene subgroup from the J558 gene family.     

Germline V genes encode viable motheaten mouse autoantibodies against thymocytes and red blood cells

Autoantibodies against thymocytes and RBC may contribute to the pathophysiology of homozygous viable motheaten (mev) autoimmune disease. Whether the production of these autoantibodies in mev mouse results from polyclonal nonspecific B cell activation or specific Ag-driven stimulation is not known. To understand the mechanisms involved in the induction of antithymocyte autoantibody response in mev mouse, we have studied the fine antigenic specificity, structure, and origin of three antithymocyte autoantibodies derived from mev splenic B cell hybridomas. Western blot analysis showed that these mAb bind to polypeptides of 33 and 105 kDa present in RBC and thymocytes, respectively. Additional specificities for the epitopes present in other polypeptides distinguished these three autoantibodies. Northern hybridization and flow microfluorimetry analysis indicated that these hybridomas are derived from the Ly1+ B cell subset. These autoreactive Ly-1 B cell hybridomas, chosen on the basis of their specificity, expressed L chain V genes from a single VK family (VK9) and VH genes from J606 and S107 families. Hybridomas UN34.11 and UN42.5 expressed the VK9 gene identical to that used by peritoneal Ly1+ B cells from various mouse strains and malignant B lymphoma cells secreting anti-mouse RBC treated with proteolytic enzyme bromelin and anti-SRBC antibodies. The third hybridoma, S2-14.2, used a VK9 gene identical to that expressed by MOPC41. None of the VK genes encoding these autoantibodies showed any somatic mutations. In the case of VH genes, the two hybridomas UN42.5 and S2-14.2 derived from two separate fusions, used identical VH genes from the J606 family. The third hybridoma UN34.11 used unmutated V11 germline VH gene, a member of the S107 family. Southern hybridizations, using oligonucleotide probes specific for CDR1 and CDR2, showed that the VH genes encoding the J606 autoantibodies were derived from a germline gene found in the 6.7-kb fragment of EcoRI-digested germline DNA. This germline VH gene is distinct from VH22.1 germline gene that codes for antigalactan antibodies. Sequence analysis of this gene showed perfect homology with the rearranged VH genes confirming the lack of somatic mutations. Thus, our data demonstrate that antithymocyte antibody response occurring in mev mouse is polyclonal and it involves Ly-1 B cells expressing unmutated germline VH and VK genes. These results indicate that antigen driven stimulation may not play an important role in the induction of anti-thymocyte antibody response in mev mouse.     

M protein (M1) of influenza virus: antigenic analysis and intracellular localization with monoclonal antibodies.

A panel of 16 monoclonal antibodies recognizing M protein (M1) of influenza virus was generated. Competition analyses resulted in localization of 14 monoclonal antibodies to three antigenic sites. Three monoclonal antibodies localized to site 1B recognized a peptide synthesized to M1 (residues 220 to 236) with enzyme-linked immunosorbent assay titers equivalent to or greater than that seen with purified M1; therefore, site 1B is located near the C terminus of M1. Sites 2 and 3 localize to the N-terminal half of M1. Antigenic variation of M proteins was seen when the monoclonal antibodies were tested against 14 strains of type A influenza viruses. Several monoclonal antibodies showed specific recognition of A/PR/8/34 and A/USSR/90/77 M proteins and little or no reactivity for all other strains tested. Immunofluorescence analysis with the monoclonal antibodies showed migration of M protein to the nucleus during the replicative cycle and demonstrated association of M protein with actin filaments in the cytoplasm. Use of a vaccinia virus recombinant containing the M-protein gene demonstrated migration of M protein to the nucleus in the absence of synthesis of gene products from other influenza virus RNA segments.     

Cooperation between the Hemagglutinin of Avian Viruses and the Matrix Protein of Human Influenza A Viruses

To analyze the compatibility of avian influenza A virus hemagglutinins (HAs) and human influenza A virus matrix (M) proteins M1 and M2, we doubly infected Madin-Darby canine kidney cells with amantadine (1-aminoadamantane hydrochloride)-resistant human viruses and amantadine-sensitive avian strains. By using antisera against the human virus HAs and amantadine, we selected reassortants containing the human virus M gene and the avian virus HA gene. In our system, high virus yields and large, well-defined plaques indicated that the avian HAs and the human M gene products could cooperate effectively; low virus yields and small, turbid plaques indicated that cooperation was poor. The M gene products are among the primary components that determine the species specificities of influenza A viruses. Therefore, our system also indicated whether the avian HA genes effectively reassorted into the genome and replaced the HA gene of the prevailing human influenza A viruses. Most of the avian HAs that we tested efficiently cooperated with the M gene products of the early human A/PR/8/34 (H1N1) virus; however, the avian HAs did not effectively cooperate with the most recently isolated human virus that we tested, A/Nanchang/933/95 (H3N2). Cooperation between the avian HAs and the M proteins of the human A/Singapore/57 (H2N2) virus was moderate. These results suggest that the currently prevailing human influenza A viruses might have lost their ability to undergo antigenic shift and therefore are unable to form new pandemic viruses that contain an avian HA, a finding that is of great interest for pandemic planning.     

Origin of the autoreactive anti-type II collagen response. II. Specificities, antibody isotypes and usage of V gene families of anti-type II collagen B cells

Autoantibodies play an important role in the pathogenesis of type II collagen-induced arthritis in mice. We have earlier reported a high frequency of cells producing anti-CII autoantibodies and a low frequency of cells producing multispecific antibodies, in regional lymph nodes 9 to 11 days after primary immunization with CII. It is shown here that anti-CII antibodies produced during primary immune response are IgG-antibodies mainly of IgG2a, IgG1 and IgG2b subclasses while IgM antibodies dominate primary responses elicited by OVA and denatured CII as analyzed with a large panel of hybridomas. Anti-CII antibodies generated during the primary response recognize at least five different epitopes on the CII molecule. The specificities of these antibodies for various epitopes result from combinational association of products encoded by genes derived from various VH and VK families and/or by the occurrence of somatic mutations. It is suggested that the primary anti-CII autoantibody response involves activation of memory B cells and is in this aspect different from the origin of "natural" autoantibodies.     

Influence of a V kappa 8 L chain transgene on endogenous rearrangements and the immune response to the HA(Sb) determinant on influenza virus

A rearranged murine V kappa 8/J kappa 5 L chain gene that codes for the L chain of most antibodies generated in the primary response of BALB/c mice to the antigenic site, Sb, of the hemagglutinin (HA) molecule of influenza virus A/PR/8/34 (PR8) has been cloned. Three transgenic lines were generated by microinjecting the gene. Lines Ga and L each contain a single copy of the transgene whereas line Gb contains three complete copies. Mice of the Ga lineage showed increased V kappa 8-specific mRNA levels only in spleen, but not in nonlymphoid organs and therefore displayed apparently normal lymphoid-specific regulation of the Ig transgene. B cell hybridomas generated from these mice were analyzed for rearrangements of endogenous V kappa genes. Greater than 90% of the C kappa alleles were retained in germ-line configuration in the Ga line, compared with only 0 to 18% in the L line. Thus, a wide variation in the frequency of endogenous rearrangements is seen among mice of different lineages using the same transgene construct. None of more than 150 hybridomas derived from LPS-stimulated splenic B cells of Ga mice exhibited HA-binding activity although they expressed the transgene and, in most cases, excluded endogenous V kappa rearrangements. In contrast, a large fraction of hybridomas isolated after primary immunization with PR8 were HA(Sb)-specific. This indicated that the transgene was functional but formed HA-specific antibodies with a more restricted set of H chains than previously hypothesized. The primary anti-HA response to immunization with PR8 was diminished in all lines compared with normal mice except for a slightly accelerated but transient burst of anti-HA antibody formation in two out of three lines (Ga and Gb). This early response in G lineage mice was largely specific for HA(Sb) and thus appeared to be composed of transgene-expressing antibodies. No differences in serum titers were observed in the secondary anti-HA responses to booster inoculation with PR8 between transgenic and normal mice.     

Allogeneic manipulation of the GAT idiotypic cascade. Immunization of C57BL/6 mice by BALB/c anti-idiotypes stimulates similar strain-specific V genes as the original antigen

Antibodies specific for the immunizing Ag (Ab1) (Id+ Ag+) and Ab3 (Id+ Ag+ or Id+ Ag-) of the (Glu60 Tyr10 Ala30) (GAT) idiotypic cascade express similar pGAT public determinants in BALB/c and C57BL/6 strains. These determinants have been shown to be dependent upon both VH and Vkappa encoded segments. The VH of the BALB/c Ab1 (germ-line gene H10) and that of the C57BL/6 Ab1 (germ-line gene V186-2) are only 75% homologous, whereas VK are much more conserved. C57BL/6 mice were immunized with BALB/c Ab2 (anti-idiotypic) antibodies and monoclonal Ab3 were derived after fusion of immunized spleen cells with the nonsecreting hybridoma cell line Sp/2.0-Ag. From 13 cell lines, five clones (four Id+ Ag- and one Id+ Ag+) were isolated and the mRNA V regions sequenced. Immunization with BALB/c anti-idiotypes elicits expression of the same or closely related C57BL/6 VH and Vkappa genes as when C57BL/6 mice were immunized with GAT, although functional VH BALB/c equivalents have been isolated in the B6 strain. Our results suggest that manipulation of the repertoire via antigenic or idiotypic stimulation both lead to the expression of different genes in different strains. They further confirm that the immune system is largely degenerate, for both idiotype expression and Ag recognition.     

Autoantibodies to thyroglobulin are encoded by diverse V-gene segments and recognize restricted epitopes.

Murine experimental autoimmune thyroiditis has been used as a model for human autoimmune thyroiditis. Experimental autoimmune thyroiditis is induced in mice by immunization with mouse thyroglobulin (Tg) in CFA. To characterize the antibodies to this autoantigen, we have studied the binding specificities and determined the nucleotide sequences of monoclonal anti-Tg antibodies. The specificities of the mAb for determinants on Tg varied extensively. Seven of 16 mAb showed reactivity to only mTg, 4 reacted to Tg from more than one species and four reacted to a variety of Ag. Many of the mAb were competitively inhibited by thyroid hormones, suggesting that they recognize the hormonogenic sites on the Tg molecule. The mAb could be divided into at least seven reactivity patterns based on reciprocal competitive inhibition studies, indicating that mTg contains at least seven antigenic regions. DNA sequence analysis of the mAb showed that a large number of V region gene segments encoded the H and L chains. No evidence for preferential use of any V region family or gene segment was found. Gene segments from the VH 7183, Q52, J558, and VH10 families were used by heavy chains, and the V kappa 1, 4, 8, 9, 19, and 21 families were used by kappa-chains. The results indicate that the antigenic epitopes on mTg elicit a very diverse autoantibody response that is derived from a large number of V region gene segments. Many of these autoantibodies show specific reactivity with mTg indicating they recognize species specific epitopes. The results suggest that clonal deletion of autoreactive Ab to certain self-epitopes may not occur.     

Specific VDJ gene combinations contribute to the specificity of memory antibodies to the phosphorylcholine hapten.

Based on their fine specificity, two groups of antibodies against the phosphorylcholine (PC) hapten have been described. Group I antibodies react predominantly with the PC moiety of the hapten and group II are directed against the entire hapten including the azophenyl spacer to the protein carrier. We have analyzed the VH gene segment utilization of hybridomas from the memory response to PC by Southern blot analysis and nucleotide sequencing of the functional VDJ rearrangements. Three main specificities of anti-PC antibodies could be distinguished. Anti-PC hybridomas with group I fine specificity utilize the VH1-DFL 16.1-JH1 rearrangement. A major portion of group II antibodies recognized the phenyl-PC part and expressed the same VH1 gene in combination with a member of the SP2 family and JH1 or JH2. The other anti-PC antibodies either used the PJ14-DFL16-JH3 rearrangement in combination with a lambda 1 L chain or a member of the VGam3.8 VH family rearranged to the DFL16.1 and the JH3 gene segments. The PJ14 and VGam3.8 V gene expressing antibodies were directed to the phenyl group and were either not or barely inhibitable by PC chloride. Thus, specific VDJ gene combinations contribute to the fine specificity of antibodies in the memory response to the PC hapten. The use of the S107, Q52, and VGam3.8. VH gene families, together with FL16.1 or SP2 D segments and JH1, JH2, or JH3 results in different fine specificities to the PC, phenyl-PC, or the azophenyl moiety of the PC hapten. These fine specificities of the memory response use V, D, and J segments of the initial T15Id+ response in combination with gene segments usually related to phenyl specificity.     

V region sequences of anti-DNA and anti-RNA autoantibodies from NZB/NZW F1 mice

The V region sequences of two anti-DNA (A52, D42) and two anti-RNA (D44, D444) autoantibodies, derived from lupus prone NZB/NZW F1 female mice, were determined by mRNA sequencing. The sequences had the following features: 1) there was no clear sequence relationship between anti-DNA and anti-RNA antibodies; 2) there were no major similarities between any of the L chain sequences and each VL gene segment belonged to a different mouse VK subgroup; 3) the H chains of the two anti-RNA antibodies showed closely related sequences of VH gene segments and very similar third complementarity determining regions (CDR3); 4) the H chains of the two anti-DNA antibodies had VH segments belonging to different VH gene families but had a unique and similar combination of D segments and junctional sequences, suggesting a common recognition element for Ag and/or for idiotypic regulation in the H chain CDR3; and 5) the VH gene segment of one anti-DNA antibody (D42) was found to be very similar to the VH gene segment of a CBA mouse hybridoma antibody (6G6) which binds to the environmental Ag phosphocholine. The three-dimensional structure of the Fv-region of the anti-DNA antibody (D42) was modeled by computer and a stretch of poly(dT), ssDNA was docked to a cleft in the antibody combining site, formed by the three H chain CDR and by CDR1 and CDR3 of the L chain. The cleft is characterized by a preponderance of arginine and tyrosine residues, lining both the walls and base of the cleft.     

Nucleotide and translated amino acid sequences of cDNA coding for the variable regions of the light and heavy chains of mouse hybridoma antibodies to blood group A and B substances

This is the first report of nucleotide and translated amino acid sequences of the variable region light (VL) and heavy (VH) chains of mouse monoclonal hybridoma anti-blood group A and B substances, the combining sites of which have been mapped. Monoclonal hybridoma anti-A and anti-B produced in BALB/c mice by immunization with A or B blood group substances, with A1 erythrocytes, and water-soluble blood group A substance or with synthetic B determinants coupled to bovine serum albumin or to O erythrocytes have been characterized immunochemically. To relate the immunochemical properties of the monoclonals to their primary structures, we have cloned and sequenced cDNAs of variable regions of light and heavy chains of two anti-A and two anti-B. The anti-A hybridomas have very similar combining site specificities and have almost identical VH sequences belonging to the J558 germ-line family, but their VL are from different germ-line VK gene families. The two anti-B hybridomas have different combining site specificities and use the same VL which differs completely from the anti-A VL; their VH are derived from different VH germ-line genes belonging to the J606 family. The results suggest that the heavy chains play a major role in determining the specificities of the antibody combining sites, with only minor contribution of VL. Additional sequence data on monoclonal antibodies of defined specificity for blood group substances are needed for further insights into the genetic and structural basis for their specificities.     

Characteristics of murine monoclonal anti-CD4. Epitope recognition, idiotype expression, and variable region gene sequence.

We have characterized a series of mouse monoclonal anti-CD4 and describe both their CD4 epitope recognition and Id expression. We also determined the V region gene sequences of these antibodies in an attempt to correlate epitope recognition and Id expression with V region sequence. All of these preparations recognize epitopes that cluster around the HIV gp120 binding site on the human CD4 molecule. However, we observed differences in epitope recognition among the anti-CD4 preparations, based on either competitive inhibition assays or functional assays, such as syncytium inhibition. Analysis of Id specificities using a polyclonal anti-Id generated against anti-Leu 3a indicated that five of the seven monoclonal anti-CD4 expressed a shared Id. Based on V region gene sequences, the V region kappa-chain (V[kappa]) from each of the seven antibodies was encoded by the V[kappa]21 gene family and expressed the J[kappa]4 gene segment. Those preparations that expressed the shared Id with anti-Leu 3a have virtually identical V[kappa] sequences, with a high degree of homology in the CDR. The VH region gene sequences of six of the seven antibodies also shared overall homology and appeared to be encoded by the J558 VH gene family. The seventh anti-CD4 VH region is encoded for by the VHGAM gene family. The majority of these antibodies used JH3 gene segment, although the JH2 and JH4 gene segments were also represented. In addition, several of these antibodies share a common sequence organization within their V-D-J joining regions that appears to involve N and P sequences to generate unique D segments. Together, these data suggest that differences in epitope recognition among the monoclonal anti-CD4 may reflect sequence variability primarily within the CDR3 region of both V[kappa] and VH. The basis for the detection of a shared Id most likely reflects the high degree of homology within the V[kappa] region sequences. In addition, these data, which are based on a limited analysis, suggest the possible restricted use of V region germ-line gene families in the secondary antibody response of BALB/c mice to specific epitopes on the human CD4 molecule.     

Primary structure of the full-size DNA copy of the influenza virus A/Kiev/59/79 (H1N1) PB1 gene protein

Nucleotide sequence of the A/Kiev/59/79 influenza virus PB1 gene is reported, thus completing the full-genome primary structure of the recombinant between the virus and laboratory strain A/PR/8/34. The parental strain A/Kiev/59/79 (H1N1) is, in turn, shown to be a natural reassortant inheriting its genes of polymerase complex (PB1, PB2, NP and, in all probability, PA) from contemporary H3N2 influenza virus strains.     

Relationship of human variable region heavy chain germ-line genes to genes encoding anti-DNA autoantibodies

In order to identify the V region genes encoding systemic lupus erythematosus (SLE)-derived anti-DNA autoantibodies, we have determined the nucleotide sequence of heavy chain mRNA from several DNA-binding immunoglobulins secreted by human hybridomas. We used the technique of cDNA primer extension for determining sequences of the VH, D, and JH gene segments of anti-DNA autoantibodies from three different primary hybridoma growths from an SLE patient and one hybridoma from a leprosy patient. Immunoglobulins from two of the SLE hybridomas expressed the same idiotype, Id-16/6, which is also expressed on immunoglobulins in sera of patients with active SLE. Their mRNA sequences showed complete homology to each other in the V, D, and J genes and more than 99% homology to the VH26 germ-line gene sequence, a member of the human VHIII gene family. The VH mRNA sequence of the third SLE hybridoma, 21/28, which was idiotypically unrelated to the other two, was 93% homologous to a different VH germ-line gene sequence, HA2, a member of the human VHI gene family. The fourth anti-DNA-producing hybridoma, 8E10, was derived from a leprosy patient of different ethnic origin than the SLE patient. It was idiotypically related to 21/28 and expressed a VH segment gene identical to that of 21/28. Hybridomas 21/28 and 8E10 shared sequence homology with the VH26 anti-DNA antibodies in the first complementarity-determining region. In addition, 21/28 shared sequence homology with the Id-16/6+ group in the region encoded by the D and J gene segments. Our findings indicate that some SLE autoantibodies are encoded by unmodified or scarcely modified VH germ-line genes that are conserved in the human population and identify two distinct VH germ-line genes that can encode segments of anti-DNA immunoglobulins.     

The predicted antigenicity of the haemagglutinin of the 1918 Spanish influenza pandemic suggests an avian origin.

In 1982 we characterized the antigenic sites of the haemagglutinin of influenza A/PR/8/34, which is an influenza strain of the H1 subtype that was isolated from humans in 1934, by studying mutants which escaped neutralization by antibody. Four antigenic sites, namely Cb, Sa, Sb and Ca, were found to be located near the tip of the trimeric haemagglutinin spike. Based on the sequence of the haemagglutinin of the 1918 Spanish influenza, we can now specify the extent of divergence of antigenic sites of the haemagglutinin during the antigenic drift of the virus between 1918 and 1934. This divergence was much more extensive (40%) than the divergence (20%) in predicted antigenic sites between the 1918 Spanish influenza and an avian H1 subtype consensus sequence. These results support the hypothesis that the human 1918 pandemic originated from an avian virus of the H1 subtype that crossed the species barrier from birds to humans and adapted to humans, presumably by mutation and/or reassortment, shortly before 1918.