# Patent

## Title
HIV envelope polynucleotides and immunogenic composition

## Abstract

Oligonucleotide sequences encoding gp120 polypeptides from breakthrough isolates of vaccine trials using MN-rgp120 and the encoded gp120 polypeptides are provided. Use of the gp120 polypeptides from one or more of the isolates in a subunit vaccine, usually together with MN-rgp120, can provide protection against HIV strains that are sufficiently different from the vaccine strain (e.g.; MN-rgp120) that the vaccine does not confer protection against those strains. Antibodies induced by the polypeptides are also provided.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a divisional of application Ser. No. 09/419,362, filed Oct. 15, 1999, now U.S. Pat. No. 6,585,979 which is a divisional of application Ser. No. 08/889,841 (now U.S. Pat. No. 6,090,392), filed Jul. 8, 1997, which claims the benefit of U.S. Provisional Application No. 60/069,891 filed Jul. 8, 1996 abandoned.


BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to HIV envelope polypeptides and vaccines containing the polypeptides.
2. Description of the Related Art
Acquired immunodeficiency syndrome (AIDS) is caused by a retrovirus identified as the human immunodeficiency virus (HIV). There have been intense efforts to develop a vaccine that induces a protective immune response based on induction of antibodies or cellular responses. Recent efforts have used subunit vaccines where an HIV protein, rather than attenuated or killed virus, is used as the immunogen in the vaccine for safety reasons. Subunit vaccines generally include gp120, the portion of the HIV envelope protein which is on the surface of the virus.
The HIV envelope protein has been extensively described, and the amino acid and nucleic acid sequences encoding HIV envelope from a number of HIV strains are known (Myers, G. et al., 1992. Human Retroviruses and AIDS. A compilation and analysis of nucleic acid and amino acid sequences. Los Alamos National Laboratory, Los Alamos, N. Mex.). The HIV envelope protein is a glycoprotein of about 160 kd (gp160) which is anchored in the membrane bilayer at its carboxyl terminal region. The N-terminal segment, gp120, protrudes into the aqueous environment surrounding the virion and the C-terminal segment, gp41, spans the membrane. Via a host-cell mediated process, gp160 is cleaved to form gp120 and the integral membrane protein gp41. As there is no covalent attachment between gp120 and gp41, free gp120 is sometimes released from the surface of virions and infected cells.
The gp120 molecule consists of a polypeptide core of 60,000 daltons which is extensively modified by N-linked glycosylation to increase the apparent molecular weight of the molecule to 120,000 daltons. The amino acid sequence of gp120 contains five relatively conserved domains interspersed with five hypervariable domains. The positions of the 18 cysteine residues in the gp120 primary sequence, and the positions of 13 of the approximately 24 N-linked glycosylation sites in the gp120 sequence are common to all gp120 sequences. The hypervariable domains contain extensive amino acid substitutions, insertions and deletions. Sequence variations in these domains result in up to 30% overall sequence variability between gp120 molecules from the various viral isolates. Despite this variation, all gp120 sequences preserve the ability of the virus to bind to the viral receptor CD4 and to interact with gp41 to induce fusion of the viral and host cell membranes.
gp120 has been the object of intensive investigation as a vaccine candidate for subunit vaccines, as the viral protein which is most likely to be accessible to immune attack. At present, clinical trials using gp120 MN strain are underway. However, to date no human vaccine trial has been of sufficient size to confirm or refute vaccine efficacy.
The development of candidate HIV-1 vaccines is burdened by the lack of in vivo or in vitro models of HIV-1 infection that accurately approximate the conditions of natural infection in humans. Several candidate HIV-1 vaccines [Berman et al.; J. Virol. 7:4464–9 (1992); Haigwood et al.; J. Virol. 66:172–82 (1992); Salmon-Ceron et al.; AIDS Res. and Human Retroviruses 11:1479–86 (1995)] have been described that elicit broadly cross-reactive antibodies able to neutralize a variety of diverse HIV-1 isolates in vitro. However, the relevance of in vitro assays to protective immunity in vivo is uncertain. Although several vaccines have provided chimpanzees with protection from challenge by homologous and heterologous strains of HIV-1, protection has not always correlated with in vitro neutralization assays carried out in T cell lines, or in lectin and cytokine activated peripheral blood mononuclear cells (PBMCs) [Berman et al.; Nature 345:622–5 (1990); Bruck et al.; Vaccine 12(12):1141–8 (1994); El-Amad et al.; AIDS 9:1313–22 (1995); Girard et al.; J. Virol. 69:6239–48 (1995); and Fulz et al; Science 256:1687–1690 (1992)]. While successful protection of chimpanzees is encouraging and has historically proved to be a reliable indicator of vaccine efficacy, the conditions of infection in all experimental models of HIV-1 infection differ significantly from natural infection in humans.
Experimental HIV-1 infection in vivo and in vitro both suffer from the limitation that the in vitro amplification of HIV-1, which is required to prepare virus stocks for in vitro or in vivo infectivity experiments, imposes a genetic selection that results in a spectrum of virus quasi-species that differ from the spectrum of variants present in the clinical specimens used to establish the culture [Kusumi et al.; J. Virol. 66:875 (1992); Meyerhans et al.; Cell 58:901–10 (1989)]. Because of these uncertainties, and even greater uncertainties related to the amount of virus transmitted, the site and cell type involved in initial replication, and the kinetics of virus dissemination, the ability of currently available in vitro or in vivo assays to reliably predict vaccine efficacy is questionable.
One of the candidate HIV-1 vaccines that have entered human clinical trials is recombinant gp120 prepared in Chinese hamster ovary (CHO) cells from the MN strain of HIV-1 (MN-rgp120) (Berman et al.; J. Virol. 7:4464–9 (1992)). To date, approximately 499 adults have participated in Phase 1 and 2 immunogenicity and safety trials of this vaccine. The data collected thus far suggest that MN-rgp120 is safe, immunogenic, and elicits high titers of neutralizing antibodies in greater than 95% of individuals immunized according to a 0, 1, and 6 month immunization schedule [Belshe et al.; JAMA 272(6):475–80 (1994); McElrath; Seminars in Cancer Biol. 6:1–11 (1995)]. However, during the course of these trials, nine vaccinees who received MN-rgp120 have become infected with HIV-1 through high risk behavior. Small trials, such as these, in populations with low rates of infection and minimally sized placebo control groups do not have sufficient statistical power to confirm or refute vaccine efficacy.
However, effective vaccines based on gp120 or another HIV protein for protection against additional strains of HIV are still being sought to prevent the spread of this disease.
DESCRIPTION OF THE BACKGROUND ART
Recombinant subunit vaccines are described in Berman et al., PCT/US91/02250 (published as number WO91/15238 on 17 Oct. 1991). See also, e.g. Hu et al., Nature 328:721–724 (1987) (vaccinia virus-HIV envelope recombinant vaccine); Arthur et al., J. Virol. 63(12): 5046–5053 (1989) (purified gp120); and Berman et al., Proc. Natl. Acad. Sci. USA 85:5200–5204 (1988) (recombinant envelope glycoprotein gp120).
Numerous sequences for gp120 are known. The sequence of gp120 from the IIIB substrain of HIV-1LAI referred to herein is that determined by Muesing et al., “Nucleic acid structure and expression of the human AIDS/lymphadenopathy retrovirus,” Nature 313:450–458 (1985). The sequences of gp120 from the NY-5, Jrcsf, Z6, Z321, and HXB2 strains of HIV-1 are listed by Myers et al., “Human Retroviruses and AIDS; A compilation and analysis of nucleic acid and amino acid sequences,” Los Alamos National Laboratory, Los Alamos, N. Mex. (1992). The Thai isolate CM244 is described by McCutchen et al., “Genetic Variants of HIV-1 in Thailand,” AIDS Res. And Human Retroviruses 8:1887–1895 (1992). The MN1984 clone is described by Gurgo et al., “Envelope sequences of two new United States HIV-1 isolates,” Virol. 164:531–536 (1988). As used herein, MN, MN-rgp120, the MN clone or isolate refers to MHGNE. The MNGNE amino acid sequence is Sequence ID NO:41.
Each of the above-described references is incorporated herein by reference in its entirety.
SUMMARY OF THE INVENTION
Oligonucleotide sequences encoding gp120 polypeptides from breakthrough isolates of vaccine trials using MN-rgp120 and the encoded gp120 polypeptides are provided. Use of the gp120 polypeptides from one or more of the isolates in a subunit vaccine, usually together with MN-rgp120, can provide protection against HIV strains that are sufficiently different from the vaccine strain (e.g.; MN-rgp120) that the vaccine does not confer protection against those strains. Antibodies induced by the polypeptides are also provided.



BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A–1G illustrate the kinetics of antibody response to MN-rgp120 in vaccinees infected with HIV-1. Sera were collected at the time points indicated and assayed for antibodies reactive with MN-rgp120 (open circles) or a synthetic peptide derived from the V3 domain of MN-rgp120 (closed circles). Arrows indicate dates of injection. Plus sign indicates the first time HIV-1 infection was detected. Shaded area indicates data collected after HIV-1 infection. Data from vaccinee C6 is shown in FIG. 1A; C8 in FIG. 1B; C7, FIG. 1C; C11, FIG. 1D; C10, FIG. 1E; C17, FIG. 1F; and C15, FIG. 1G.
FIGS. 2A–2G illustrate the kinetics of CD4 blocking antibody response in vaccinees infected with HIV-1. Sera were collected at the time points indicated and assayed for antibodies able to block the binding of [125I]-labeled MN-rgp120 to cell surface CD4. Arrows indicate dates of injection. Plus sign indicates the first time HIV-1 infection was detected. Shaded area indicates data collected after HIV-1 infection. Data from vaccinee C6 is shown in FIG. 2A; C8 in FIG. 2B; C7, FIG. 2C; C11, FIG. 2D; C10, FIG. 2E; C17, FIG. 2F; and C15, FIG. 2G.
FIGS. 3A–3J illustrate predicted amino acid sequences of envelope glycoproteins (gp120) from breakthrough viruses. Proviral DNA sequences were amplified by PCR from PBMCs and cloned into the PRK5 expression plasmid. Two clones from each infected vaccinnee were sequenced from double stranded plasmid DNA. Sequence numbering is with reference to the initiator methionine residue of gp120. For the purpose of comparison, the sequences shown bein at amino acid 12 of the mature, fully processed, envelope glycoproteins (corresponding to position 41 of the gp120 open reading frame). Shaded areas indicate sequences at neutralizing epitopes, dark boxes indicate polymorphisms thought to be important for the binding of virus neutralizing Mabs reactive with MN-rgp120. Conserved (C) regions and variable (V) regions are indicated above the sequences. Boxes indicate sequence homologies and polymorphisms. The sequences of the clones shown (i.e., C6.1–C17.3) are found in the Sequence Listing in SEQ ID NOs: 2, 5, 8, 10, 12, 16, 19, 23, 25, 28, 31, 33, 36, and 39, respectively. The sequence of MNGNE appears in the Sequence Listing as SEQ ID NO: 41.
FIG. 4 illustrates immunoprecipitation of recombinant gp120 prepared from breakthrough viruses. Recombinant gp120s from the seven breakthrough viruses were prepared by transient transfection of 293s cells. Cells were metabolically labeled with 35S methionine and growth conditioned cell culture supernatants were immunoprecipitated with polyclonal antisera to MN-rgp120. Immunoprecipitates were resolved by SDS-PAGE and visualized by autoradiography. C8 lanes a and b correspond to clones C8.3 and C8.6; C6 lanes a and b correspond to clones C6.1 and C6.5; C7 lanes a and b correspond to clones C7.2 and C7.10; C17 lanes a and b correspond to C17.1 and C17.3; C11 lanes a and b correspond to clones C11.5 and C11.7; C10 lanes a and b correspond to clones C10.5 and C10.7; C15 lanes a and b correspond to clones C15.2 and C15.3.
FIGS. 5A–5D illustrate binding of monoclonal antibodies to recombinant gp120 from breakthrough viruses. Growth-conditioned cell culture supernatants were collected from 293s cells transiently transfected with plasmids directing the expression of breakthrough virus envelope glycoproteins. The relative rgp120 concentrations were determined by ELISA using MAb 5B6 specific for the HSV-1 glycoprotein D flag epitope at the amino terminus of all of the rgp120 variants described herein. The resulting rgp120 preparations were captured onto wells of microtiter plates coated with a polyclonal antibody specific for a conserved sequence in the C-terminus of gp120. The binding of virus neutralizing monoclonal antibodies reactive with gp120 was determined by ELISA. FIG. 5A, binding by MAb (5B6) specific for the HSV-1 glycoprotein D flag epitope; FIG. 5B, binding by MAb (1034) against the V3 domain of MN-rgp120; FIG. 5C binding by MAb (50.1) raised against a synthetic peptide corresponding to the V3 domain of MN-rgp120; FIG. 5D, binding by a human MAb (15e) known to block the binding of gp120 to CD4.
FIG. 6 depicts the mature envelope glycoprotein (gp120) from the MN clone of the MN strain of HIV-1 (SEQ. ID NO: 41). Hypervariable domains are indicated in bold, and the V and C regions are indicated (according to Modrow et al., J. Virology 61(2):570 (1987). Potential glycosylation sites are marked with a (*).



DETAILED DESCRIPTION OF THE INVENTION
The present invention provides gp120 polypeptides from breakthrough isolates of HIV vaccine trials. Novel oligonucleotide sequences encoding gp120 from breakthrough isolates which can be used to express gp120 are also provided. Use of gp120 polypeptides from one or more of the isolates in a subunit vaccine, usually together with MN-rgp120, can provide protection against HIV strains that are sufficiently different from the vaccine strain (e.g.; MN-rgp120) that the vaccine does not confer protection against those strains.
In one embodiment, the vaccine is based on the use of the MN-rgp120 polypeptide (Sequence ID NO: 41) and gp120 polypeptides from MN-like viruses that include neutralizing epitopes that are not present in the initial vaccine strain, and are sufficiently different from those of the vaccine strain, to have been able to cause HIV-1 infections in MN-rgp120 vaccinated individuals (i.e.; to result in breakthrough infections). Use of the initial vaccine strain empirically determines the viruses present in the population that contain additional neutralizing epitopes sufficiently different from those of the vaccine strain to escape protection induced by the vaccine strain. Use of an initial representative gp120 polypeptide in a vaccine acts as a sieve so that viruses that are not effectively protected against by the vaccine strain breakthrough the vaccine, empirically resulting in determination of additional strains in a given geographic region that are not is protected against by the initial vaccine strain. Use of gp120 from those breakthrough isolates complements the vaccine isolate by providing additional neutralizing epitopes not present in the initial vaccine strain, therefore creating a more complete vaccine that confers protection against multiple different virus strains in the region.
Prior HIV-1 vaccine strategies were based on selection of appropriate candidate vaccine polypeptides based on homology alignment studies. However, since some of the neutralizing epitopes are conformation-dependent and the location of all of these epitopes is not known, this approach necessarily cannot determine all of the neutralizing epitopes that should be included in a vaccine for a particular region. In contrast, the present approach uses a selected representative strain and empirically determines strains that are sufficiently different and therefore breakthrough the barrier of protection provided by the initial vaccination program. Those strains can be included in the vaccine to confer more complete protection from HIV strains in the region. In addition, those strains can be used alone to confer protection against the breakthrough virus.
In another embodiment, the invention comprises a vaccine containing a first HIV gp120 polypeptide sequence and a breakthrough isolate HIV gp120 polypeptide sequence from a vaccinee vaccinated with a vaccine including the first HIV gp120 polypeptide sequence, the HIV gp120 polypeptide sequences being in a suitable carrier. Fragments of one or both HIV gp120 polypeptide sequences can be substituted for one or both of the corresponding HIV gp120 polypeptide sequences.
Preferably, the first gp120 polypeptide sequence contains neutralizing epitopes found in one or more gp120 polypeptides present in isolates from the geographical region where the initial vaccine (i.e., the vaccine that gives rise to the breakthrough isolate) is administered. More preferably, the first gp120 polypeptide sequence contains at least one of the more common neutralizing epitopes for the region, and most preferably the first gp120 polypeptide sequence contains at least one of the three most common neutralizing epitopes.
gp120 polypeptide sequences suitable for use as the first gp120 polypeptide sequence include gp120 MN, the Thai isolate CM244 sequence (hereinafter “gp120 CM244”), gp120 MN-GNE6 (Sequence ID NOs: 43 and 44; also known in the art as “gp120 GNE6”), and gp120 MIN-GNE8 (Sequence ID NO: 46; also known in the art as “gp120 GNE8”), and the like. gp120 MN, gp120 MN-GNE6, and gp120 MN-GNE8 are especially preferred for use as the first gp120 polypeptide sequence in initial vaccines for North America. gp120 CM244 is especially preferred for use as the first gp120 polypeptide sequence in initial vaccines for Thailand.
In a variation of this embodiment, the vaccine includes two different (i.e., first and second) gp120 polypeptide sequences, or fragments thereof, in combination with a breakthrough isolate HIV gp120 polypeptide sequence. The latter can be from a vaccinee vaccinated with either or both of the first and second HIV gp120 polypeptide sequences.
Exemplary vaccines include those containing combinations of gp120 MN, gp120 CM244, gp120 MN-GNE6 (Sequence ID NOs: 43 and 44), and gp120 MN-GNE 8 (Sequence ID NO: 46). Combinations of gp120 MN and gp120 CM244 or gp120 MN-GNE8 (Sequence ID NO: 46) with a breakthrough isolate HIV gp120polypeptide sequence are especially preferred.
In vaccines containing gp120 MN, the breakthrough isolate HIV gp120 polypeptide sequence can be an HIV gp120 polypeptide sequence selected from the group consisting of Sequence ID NOs: 2, 5, 8, 10, 12, 16, 19, 23, 25, 28, 31, 33, 36, and 39, and fragments thereof.
The term “subunit vaccine” is used herein, as in the art, to refer to a viral vaccine that does not contain virus, but rather contains one or more viral proteins or fragments of viral proteins. As used herein, the term “multivalent”, means that the vaccine contains gp120 from at least two HIV isolates having different amino acid sequences.
The term “breakthrough isolate” or “breakthrough virus” is used herein, as in the art, to refer to a virus isolated from a vaccinee.
The terms “amino acid sequence”, “polypeptide sequence”, and “polypeptide” are used interchangeably herein as in the art, as are the terms “nucleic acid sequence”, “nucleotide sequence”, and “oligonucleotide”.
Polypeptides from Breakthrough Isolates
The gp120 polypeptides of this invention correspond to the amino acid sequences of seven breakthrough isolates which are illustrated below in Table 1. A polypeptide of this invention includes an HIV gp120 amino acid sequence illustrated in Table 1 (Sequence ID NOs: 1, 4, 7, 9, 11, 15, 18, 22, 24, 27, 30, 32, 35, and 38) and fragments thereof. The polypeptides of this invention can include fused sequences from two or more HIV gp120 or gp160 amino acid sequences.
The polypeptide can also be joined to another viral protein, such as a flag epitope amino acid sequence. The term “flag epitope” is used herein, as in the art, to denote an amino acid sequence that includes an epitope recognized by a monoclonal antibody. Flag epitopes facilitate using single monoclonal antibody affinity purification of a plurality of different recombinant proteins, each having the flag epitope recognized by the monoclonal antibody. Numerous amino acid sequences can function as flag epitopes. The N-terminal sequences of Herpes Simplex Virus Type 1 (HSV-1) glycoprotein D (gD-1) is conveniently used as the flag epitope and its use is described in detail in the examples. The flag epitope is conveniently fused to the N terminus of the HIV gp120 polypeptide sequence. Alternatively, however, monoclonal antibodies that recognize neutralizing epitopes in the rgp120 sequences can be used to affinity purify the amino acid sequences, and a flag epitope can be omitted.
In addition, various signal sequences can be joined to a polypeptide of this invention. Although rgp120 is secreted to some extent in HIV cultures, the amount of the envelope glycoprotein released from (secreted by) the host cells varies widely from strain to strain. Various signal sequences can be introduced into the polypeptide by joining a nucleotide sequence encoding the signal sequence to the nucleotide sequence encoding the rgp120 to facilitate secretion of rgp120 from the cells. For example, Chiron HIV gp120 polypeptides include a signal sequence from tissue plasminogen activator (TPA) that provides good secretion of rgp120. Additional signal sequences are well known and include the N-terminal domain of murine leukemia virus surface protein gp70 described by Kayman et al., J. Virol. 68:400–410 (1984).
Table 1 illustrates the nucleotide and deduced amino acid sequences for two clones of each the seven breakthrough isolates of this invention. The clones are: C6.1; C6.5; C8.3; C8.6; C15.2; C15.3; C7.2; C7.10; C11.5; C11.7; C10.5; C10.7; C17.1; and C17.3. These sequence are SEQ. ID. NOs: 1–40. The amino acid sequence for MN and the nucleotide and deduced amino acid sequences for MN-GNE6 and MN-GNE8 are illustrated in the sequence listing hereinafter. In the listing for MIN-GNE6, a stop codon appears at amino acid residue position 51. This stop codon can be replaced with a codon encoding the corresponding amino acid from MN or MN-GNE8 or another isolate.








TABLE 1







CLONE C6.1










     GGG GTA CCT GTG TGG AAG GAA GCA ACC ACC ACT CTA
36



     Gly Val Pro Val Trp Lys Glu Ala Thr Thr Thr Leu


      1               5                  10


 


TTT TGT GCA TCA GAT GCT AAA GCA TAT GAC ACA GAG GTG
75


Phe Cys Ala Ser Asp Ala Lys Ala Tyr Asp Thr Glu Val


          15                  20                  25


 


CAT AAT GTT TGG GCC ACA CAT GCT TGT GTA CCC ACA GAC
114


His Asn Val Trp Ala Thr His Ala Cys Val Pro Thr Asp


                 30                  35


 


CCA AAC CCA CAA GAA ATG GTA TTG GAA AAT GTG ACA GAA
153


Pro Asn Pro Gln Glu Met Val Leu Glu Asn Val Thr Glu


     40                  45                  50


 


GAT TTT AAC ATG TGG AAA AAT GAC ATG GTA GAA CAG ATG
192


Asp Phe Asn Met Trp Lys Asn Asp Met Val Glu Gln Met


             55                  60


 


CAT GAG GAT ATA ATC AGT TTA TGG GAT CAA AGC CTA AAA
231


His Glu Asp Ile Ile Ser Leu Trp Asp Gln Ser Leu Lys


 65                  70                  75


 


CCA TGT GTA AAA TTA ACC CCA CTC TGT ATT ACT TTA AAT
270


Pro Cys Val Lys Leu Thr Pro Leu Cys Ile Thr Leu Asn


         80                  85                  90


 


TGC ACC AAT TGG AAG AAG AAT GAT ACT AAA ACT AAT AGT
309


Cys Thr Asn Trp Lys Lys Asn Asp Thr Lys Thr Asn Ser


                 95                 100


 


AGT AGT ACT ACA ACT AAT AAT AGT AGT GCT ACA GCT AAT
348


Ser Ser Thr Thr Thr Asn Asn Ser Ser Ala Thr Ala Asn


    105                 110                 115


 


AGT AGT AGT ACT ACA ACT AAT AGT AGT TGG GGA GAG ATA
387


Ser Ser Ser Thr Thr Thr Asn Ser Ser Trp Gly Glu Ile


            120                 125


 


AAG GAG GGA GAA ATA AAG AAC TGC TCT TTC AAT ATC ACC
426


Lys Glu Gly Glu Ile Lys Asn Cys Ser Phe Asn Ile Thr


130                 135                 140


 


ACA AGC ATA AGA GAC AAG GTG AAG AAA GAA TAT GCA CTT
465


Thr Ser Ile Arg Asp Lys Val Lys Lys Glu Tyr Ala Leu


        145                 150                 155


 


TTT TAT AGC CTT GAT GTA GTA CCA ATA GAA AAT GAT AAT
504


Phe Tyr Ser Leu Asp Val Val Pro Ile Glu Asn Asp Asn


                160                 165


 


ACT AGC TAT AGG TTG AGA AGT TGT AAC ACC TCA GTC ATT
543


Thr Ser Tyr Arg Leu Arg Ser Cys Asn Thr Ser Val Ile


    170                 175                 180


 


ACA CAA GCC TGT CCA AAG GTA ACT TTT GAG CCA ATT CCC
582


Thr Gln Ala Cys Pro Lys Val Thr Phe Glu Pro Ile Pro


            185                 190


 


ATA CAT TAT TGT ACC CCG GCT GGT TTT GCG ATT CTG AAG
621


Ile His Tyr Cys Thr Pro Ala Gly Phe Ala Ile Leu Lys


195                 200                 205


 


TGT AGA GAT AAA AAG TTC AAT GGA ACA GGA CCA TGC AAA
660


Cys Arg Asp Lys Lys Phe Asn Gly Thr Gly Pro Cys Lys


        210                 215                 220


 


AAT GTT AGC ACA GTA CAA TGT GCA CAT GGA ATT AAG CCA
699


Asn Val Ser Thr Val Gln Cys Ala His Gly Ile Lys Pro


                225                 230


 


GTA GTG TCA ACT CAA CTG CTG TTA AAT GGC AGC CTA GCA
738


Val Val Ser Thr Gln Leu Leu Leu Asn Gly Ser Leu Ala


    235                 240                 245


 


GAA GAA GAG GTA ATA ATT AGA TCT GCC AAT TTC TCA AAC
777


Glu Glu Glu Val Ile Ile Arg Ser Ala Asn Phe Ser Asn


            250                 255


 


AAT GCT AAA ATC ATA ATA GTA CAG TTG AGG GAA CCT GTA
816


Asn Ala Lys Ile Ile Ile Val Gln Leu Arg Glu Pro Val


260                 265                 270


 


GAA ATT AAT TGT ACA AGA CCC AGC AAC AAT ACA ATA AAA
855


Glu Ile Asn Cys Thr Arg Pro Ser Asn Asn Thr Ile Lys


        275                 280                 285


 


GGT ATA CAC ATA GGA CCA GGG AGA GCA TTT TAT GCA ACA
894


Gly Ile His Ile Gly Pro Gly Arg Ala Phe Tyr Ala Thr


                290                 295


 


GGA GAC ATA CGA GGA GAT ATA AGA CAA GCA CAT TGT AAC
933


Gly Asp Ile Arg Gly Asp Ile Ary Gln Ala His Cys Asn


    300                 305                 310


 


ATT AGT GGA GCA AAA TGG AAT AAC ACT TTA AAG AAG GTA
972


Ile Ser Gly Ala Lys Trp Asn Asn Thr Leu Lys Lys Val


            315                 320


 


GTT AAA AAA TTA AAA GAA CAA TTT CCA AAT AAA ACA ATA
1011


Val Lys Lys Leu Lys Glu Gln Phe Pro Asn Lys Thr Ile


325                 330                 335


 


GTC TTT AAC CAT TCC TCA GGA GGG GAC CCA GAA ATT GTA
1050


Val Phe Asn His Ser Ser Gly Gly Asp Pro Glu Ile Val


        340                 345                 350


 


ATG CAC AGT TTT AAT TGT CAA GGG GAA TTT TTC TAC TGT
1089


Met His Ser Phe Asn Cys Gln Gly Glu Phe Phe Tyr Cys


                355                 360


 


AAT ACA ACA AAG CTG TTT AAT AGT ACT TGG AAT GAT ACT
1128


Asn Thr Thr Lys Leu Phe Asn Ser Thr Trp Asn Asp Thr


    365                 370                 375


 


ACA GAG TCA AAT AAC AAT GAT AGT ACT ATT ACA CTC CCA
1167


Thr Glu Ser Asn Asn Asn Asp Ser Thr Ile Thr Leu Pro


            380                 385


 


TGC AGA ATA AAA CAA ATT ATA AAC ATG TGG CAG GAA ATA
1206


Cys Arg Ile Lys Gln Ile Ile Asn Met Trp Gln Glu Ile


390                 395                 400


 


GGA AAA GCA ATG TAT GCC CCT CCC ACC AGA GGA GAA ATT
1245


Gly Lys Ala Met Tyr Ala Pro Pro Thr Arg Gly Glu Ile


        405                 410                 415


 


AAA TGT TCA TCA AAT ATT ACA GGA CTA CTG TTA ATA AGA
1284


Lys Cys Ser Ser Asn Ile Thr Gly Leu Leu Leu Ile Arg


                420                 425


 


GAT GGT GGT ATT AAC ACT AGC GAT GCC ACC GAG ACC TTC
1323


Asp Gly Gly Ile Asn Thr Ser Asp Ala Thr Glu Thr Phe


    430                 435                 440


 


AGA CCG GGA GGA GGA GAT ATG A00 GAC AAT TGG AGA AGT
1362


Arg Pro Gly Gly Gly Asp Met Arg Asp Asn Trp Arg Ser


            445                 450


 


GAA TTA TAT AAA TAT AAA GTA GTG AAA ATT GAG CCA TTA
1401


Glu Leu Tyr Lys Tyr Lys Val Val Lys Ile Glu Pro Leu


455                 460                 465


 


GGA GTA GCA CCC ACC AAG GCA AAG AGA AGA GTG GTG CAG
1440


Gly Val Ala Pro Thr Lys Ala Lys Arg Arg Val Val Gln


        470                 475                 480


 


AGA GAA AAA AGA GCA GTA ACA CTA GGA GCT ATG TTC CTT
1479


Arg Glu Lys Arg Ala Val Thr Leu Gly Ala Met Phe Leu


                485                 490


 


GGG TTC TTA GGA GCA TAA AGC TTC 1503


Gly Phe Leu Gly Ala Xaa Ser Phe


    495                 500 501


 








CLONE C6.5










    GGG GTA CCT GTA TGG AAA GAA GCA ACC ACC ACT CTA
36



    Gly Val Pro Val Trp Lys Glu Ala Thr Thr Thr Leu


       1               5                  10


 


TTT TGT GCA TCA GAT GCT AAA GCA TAT GAC ACA GAG GTG
75


Phe Cys Ala Ser Asp Ala Lys Ala Tyr Asp Thr Glu Val


         15                  20                  25


 


CAT AAT GTT TGG GCC ACA CAT GCT TGT GTA CCC ACA GAC
114


His Asn Val Trp Ala Thr His Ala Cys Val Pro Thr Asp


                 30                 35


 


CCA AAC CCA CAA GAA ATG GTA TTG GAA AAT GTG ACA GAA
153


Pro Asn Pro Gln Glu Met Val Leu Glu Asn Val Thr Glu


     40                  45                  50


 


GAT TTT AAC ATG TGG AAA AAT GAC ATG GTA GAA CAG ATG
192


Asp Phe Asn Met Trp Lys Asn Asp Met Val Glu Gln Met


             55                  60


 


CAT GAG ANT ATA ATC AGT TTA TGG GAT CAA AGC CTA AAA
231


His Glu Xaa Ile Ile Ser Leu Trp Asp Gln Ser Leu Lys


 65                  70                  75


 


CCA TGT GTA AAA TTA ACC CCA CTC TGT ATT ACT TTA AAT
270


Pro Cys Val Lys Leu Thr Pro Leu Cys Ile Thr Leu Asn


        80                  85                  90


 


TGC ACC AAT TGG AAG GAG AAT GAT ACT AAA ACT AAT AGT
309


Cys Thr Asn Trp Lys Glu Asn Asp Thr Lys Thr Asn Ser


                 95                 100


 


AGT AGT ACT ACA ACT AAT AAT AGT AGT GCT ACA GCT AAT
348


Ser Ser Thr Thr Thr Asn Asn Ser Ser Ala Thr Ala Asn


    105                 110                 115


 


AGT AGT AGT ACT ACA ACT AAT AGT AGT TGG GGA GAG ATA
387


Ser Ser Ser Thr Thr Thr Asn Ser Ser Trp Gly Glu Ile


            120                 125


 


AAG GAG GGA GAA ATA AAG AAC TGC TCT TTC AAT ATC ACC
426


Lys Glu Gly Glu Ile Lys Asn Cys Ser Phe Asn Ile Thr


130                 135                 140


 


ACA GGC ATA AGA GAC AAG GTG AAG AAA GAA TAT GCA CTT
465


Thr Gly Ile Arg Asp Lys Val Lys Lys Glu Tyr Ala Leu


        145                 150                 155


 


TTT TAT AGC CTT GAT GTA GTA CCA ATA GAA AAT GAT AAT
504


Phe Tyr Ser Leu Asp Val Val Pro Ile Glu Asn Asp Asn


                160                 165


 


ACT AGC TAT AGG TTG AGA AGT TGT AAC ACC TCA GTC ATT
543


Thr Ser Tyr Arg Leu Arg Ser Cys Asn Thr Ser Val Ile


    170                 175                 180


 


ACA CAA GCC TGT CCA AAG GTA ACT TTT GAG CCA ATT CCC
582


Thr Gln Ala Cys Pro Lys Val Thr Phe Glu Pro Ile Pro


            185                 190


 


ATA CAT TAT TGT ACC CCG GCT GGT TTT GCG ATT CTG AAG
621


Ile His Tyr Cys Thr Pro Ala Gly Phe Ala Ile Leu Lys


195                 200                 205


 


TGT AAA GAT AAA AAG TTC AAT GGA ACA GGA CCA TGC AAA
660


Cys Lys Asp Lys Lys Phe Asn Gly Thr Gly Pro Cys Lys


        210                 215                 220


 


AAT GTT AGC ACA GTA CAA TGT ACA CAT GGA ATT AAG CCA
699


Asn Val Ser Thr Val Gln Cys Thr His Gly Ile Lys Pro


                225                 230


 


GTA GTG TCA ACT CAA CTG CTG TTA AAT GGC AGC CTA GCA
738


Val Val Ser Thr Gln Leu Leu Leu Asn Gly Ser Leu Ala


    235                 240                 245


 


GAA GAA GAG GTA ATA ATT AGA TCT GCC AAT TTC TCA AAC
777


Glu Glu Glu Val Ile Ile Arg Ser Ala Asn Phe Ser Asn


            250                 255


 


AAT GCT AAA ATC ATA ATA GTA CAG TTG AAG GAA CCT GTA
816


Asn Ala Lys Ile Ile Ile Val Gln Leu Lys Glu Pro Val


260                 265                 27


 


GAA ATT AAT TGT ACA AGA CCC AGC AAC AAT ACA ATA AAA
855


Glu Ile Asn Cys Thr Arg Pro Ser Asn Asn Thr Ile Lys


        275                 280                 285


 


GGT ATA CAC ATA GGA CCA GGG AGA GCA TTT TAT GCA ACA
894


Gly Ile His Ile Gly Pro Gly Arg Ala Phe Tyr Ala Thr


                290                 295


 


GGA GAC ATA CGA GGA GAT ATA AGA CAA GCA CAT TGT AAC
933


Gly Asp Ile Arg Gly Asp Ile Arg Gln Ala His Cys Asn


    300                 305                 310


 


ATT AGT GGA GCA AAA TGG AAT AAC ACT TTA AAG AAG GTA
972


Ile Ser Gly Ala Lys Trp Asn Asn Thr Leu Lys Lys Val


            315                 320


 


GTT ATA AAA TTA AAA GAA CAA TTT CCA AAT AAA ACA ATA
1011


Val Ile Lys Leu Lys Glu Gln Phe Pro Asn Lys Thr Ile


325                 330                 335


 


GTC TTT AAC CAT TCC TCA GGA GGG GAC CCA GAA ATT GTA
1050


Val Phe Asn His Ser Ser Gly Gly Asp Pro Glu Ile Val


        340                 345                 350


 


ATG CAC AGT TTT AAT TGT CAA GGG GAA TTT TTC TAC TGT
1089


Met His Ser Phe Asn Cys Gln Gly Glu Phe Phe Tyr Cys


                355                 360


 


AAT ACA ACG AAG CTG TTT AAT AGT ACT TGG AAT GAT ACT
1128


Asn Thr Thr Lys Leu Phe Asn Ser Thr Trp Asn Asp Thr


    365                 370                 375


 


ACA GAG TCA AAT AAC AAT GAT AGT ACT ATT ACA CTC CCA
1167


Thr Glu Ser Asn Asn Asn Asp Ser Thr Ile Thr Leu Pro


            380                 385


 


TGC AGA ATA AAA CAA ATT ATA AAC ATG TGG CAG GAA GTA
1206


Cys Arg Ile Lys Gln Ile Ile Asn Met Trp Gln Glu Val


390                 395                 400


 


GGA AAA GCA ATG TAT GCC CCT CCC ATC AGA GGA GAA ATT
1245


Gly Lys Ala Met Tyr Ala Pro Pro Ile Arg Gly Glu Ile


        405                 410                 415


 


AAA TGT TCA TCA AAT ATT ACA GGA CTA CTG TTA ACA AGA
1284


Lys Cys Ser Ser Asn Ile Thr Gly Leu Leu Leu Thr Arg


                420                 425


 


GAT GGT GGT ATT AAC ACT AGC GAT GCC ACC GAG ACC TTC
1323


Asp Gly Gly Ile Asn Thr Ser Asp Ala Thr Glu Thr Phe


    430                 435                 440


 


AGA CCG GGA GGA GGA GAT ATG AGG GAC AAT TGG AGA AGT
1362


Arg Pro Gly Gly Gly Asp Met Arg Asp Asn Trp Arg Ser


            445                 450


 


GAA TTA TAT AAA TAT AAA GTA GTG AAA ATT GAG CCA TTA
1401


Glu Leu Tyr Lys Tyr Lys Val Val Lys Ile Glu Pro Leu


455                 460                 465


 


GGA GTA GCA CCC ACC AAG GCA AAG AGA AGA GTG GTG CAG
1440


Gly Val Ala Pro Thr Lys Ala Lys Arg Arg Val Val Gln


        470                 475                 480


 


AGA GAA AAA AGA GCA GTA ACA CTA GGA GCT ATG TTC CTT
1479


Arg Glu Lys Arg Ala Val Thr Leu Gly Ala Met Phe Leu


                485                 490


 


GGG TTC TTG GGA GCA TAA AGC TTC 1503


Gly Phe Leu Gly Ala Xaa Ser Phe


    495                 500 501


 








CLONE C8.3










G   GTA CCT GTA TGG AAA GAA GCA ACC ACC ACT CTA TTT
37



    Val Pro Val Trp Lys Glu Ala Thr Thr Thr Leu Phe


      1               5                  10


 


TGT GCA TCA GAT GCT AAA GCA TAT GAT ACA GAG GTA CAT
76


Cys Ala Ser Asp Ala Lys Ala Tyr Asp Thr Glu Val His


         15                  20                  25


 


AAT GTT TGG GCT ACA CAT GCC TGT GTA CCC ACA GAC CCC
115


Asn Val Trp Ala Thr His Ala Cys Val Pro Thr Asp Pro


                 30                  35


 


AAC CCA CAA GAA GTA GTA TTG GAA AAT GTA ACA GAA AAT
154


Asn Pro Gln Glu Val Val Leu Glu Asn Val Thr Glu Asn


     40                  45                  50


 


TTT AAC ATG TGG AAA AAT AAC ATG GTA GAA CAG ATG CAT
193


Phe Asn Met Trp Lys Asn Asn Met Val Glu Gln Met His


             55                  60


 


GAG GAT ATA ATC AGT TTA TGG GAT CAA AGT CTA AAG CCA
232


Glu Asp Ile Ile Ser Leu Trp Asp Gln Ser Leu Lys Pro


 65                  70                 75


 


TGT GTA AAA TTA ACC CCA CTC TGT GTT ACT TTA AAT TGC
271


Cys Val Lys Leu Thr Pro Leu Cys Val Thr Leu Asn Cys


         80                  85                  90


 


ACT AAT TTG GAG AAT GCT AAT AAT ACC GAG AAT GCT AAT
310


Thr Asn Leu Glu Asn Ala Asn Asn Thr Glu Asn Ala Asn


                 95                 100


 


AAT ACC AAT AAT TAT ACC TTG GGG ATG GAG AGA GGT GAA
349


Asn Thr Asn Asn Tyr Thr Leu Gly Met Glu Arg Gly Glu


    105                 110                 115


 


ATA AAA AAC TGC TCT TTC AAT ATC ACC ACA AGC TTA AGA
388


Ile Lys Asn Cys Ser Phe Asn Ile Thr Thr Ser Leu Arg


            120                 125


 


GAT AAG GTG AAA AAA GAA TAT GCA TTG TTT TAT AAA CTT
427


Asp Lys Val Lys Lys Glu Tyr Ala Leu Phe Tyr Lys Leu


130                 135                 140


 


GAT GTA GTA CAA ATA GAT AAT AGT ACC AAC TAT AGG CTG
466


Asp Val Val Gln Ile Asp Asn Ser Thr Asn Tyr Arg Leu


        145                 150                 155


 


ATA AGT TGT AAT ACC TCA OTC ATT ACA CAG GCC TGT CCA
505


Ile Ser Cys Asn Thr Ser Val Ile Thr Gln Ala Cys Pro


                160                 165


 


AAG GTA TCC TTT GAG CTA ATT CCC ATA CAT TAT TGT GCC
544


Lys Val Ser Phe Glu Leu Ile Pro Ile His Tyr Cys Ala


    170                 175                 180


 


CCG GCT GCT TTT GCG ATT CTA AAG TGT AAA GAT AAG AAG
583


Pro Ala Gly Phe Ala Ile Leu Lys Cys Lys Asp Lys Lys


            185                 190


 


TTC AAT GGA ACA GGA CCA TGT AAA AAT OTC AGC ACA GTA
622


Phe Asn Gly Thr Gly Pro Cys Lys Asn Val Ser Thr Val


195                 200                 205


 


CAA TGT ACA CAT GGA ATT AOA CCA GTA GTA TCA ACT CAA
661


Gln Cys Thr His Gly Ile Arg Pro Val Val Ser Thr Gln


        210                 215                 220


 


CTA CTG TTA AAT GGC AGT CTA GCA GAA GAA GAG ATA GTA
700


Leu Leu Leu Asn Gly Ser Leu Ala Glu Glu Glu Ile Val


                225                 230


 


ATT AGA TCT GAA AAT ATC ACA GAC AAT GCT AAA ACC ATA
739


Ile Arg Ser Glu Asn Ile Thr Asp Asn Ala Lys Thr Ile


    235                 240                 245


 


ATA GTG CAG CTA AAT GAA TCT ATA GTG ATT AAT TGT ACA
778


Ile Val Gln Leu Asn Glu Ser Ile Val Ile Asn Cys Thr


            250                 255


 


AGA CCC AAT AAC AAC ACA AGA AAA AGT ATA AAT ATA GGA
817


Arg Pro Asn Asn Asn Thr Arg Lys Ser Ile Asn Ile Gly


260                 265                 270


 


CCA GGG AGA GCA TTC TAT ACA ACA GGA GAC ATA ATA GGA
856


Pro Gly Arg Ala Phe Tyr Thr Thr Gly Asp Ile Ile Gly


        275                 280                 285


 


GAT ATA AGA CAA GCA CAT TGT AAC CTT AGT AAA ACA CAA
895


Asp Ile Arg Gln Ala His Cys Asn Leu Ser Lys Thr Gln


                290                 295


 


TGG GAA AAA ACG TTA AGA CAG ATA GCT ATA AAA TTA GAA
934


Trp Glu Lys Thr Leu Arg Gln Ile Ala Ile Lys Leu Glu


    300                 305                 310


 


GAA AAA TTT AAG AAT AAA ACA ATA GCC TTT AAT AAA TCC
973


Glu Lys Phe Lys Asn Lys Thr Ile Ala Phe Asn Lys Ser


            315                 320


 


TCA GGA GGG GAC CCA GAA ATT GTA ATG CAC AGT TTT AAT
1012


Ser Gly Gly Asp Pro Glu Ile Val Met His Ser Phe Asn


325                 330                 335


 


TGT GGA GGG GAA TTT TTC TAC TGT AAT ACA ACA AAA CTG
1051


Cys Gly Gly Glu Phe Phe Tyr Cys Asn Thr Thr Lys Leu


        340                 345                 350


 


TTT AAT AGT ACC TGG AAT TTA ACA CAA CCG TTT AGT AAT
1090


Phe Asn Ser Thr Trp Asn Leu Thr Gln Pro Phe Ser Asn


                355                 360


 


ACC GGG AAT CGT ACT GAA GAG TTA AAT ATT ACA CTC CCA
1129


Thr Gly Asn Arg Thr Glu Glu Leu Asn Ile Thr Leu Pro


    365                 370                 375


 


TGG AGA ATA AAA CAA ATC ATA AAC TTG TGG CAG GAA GTA
1168


Cys Arg Ile Lys Gln Ile Ile Asn Leu Trp Gln Glu Val


            380                 385


 


GGC AAA GCA ATG TAT GCC CCT CCC ATC AGA GGA CAA ATT
1207


Gly Lys Ala Met Tyr Ala Pro Pro Ile Arg Gly Gln Ile


390                 395                 400


 


AGA TGT TCA TCA AAT ATT ACA GGG CTA CTA TTA ACA AGA
1246


Arg Cys Ser Ser Asn Ile Thr Gly Leu Leu Leu Thr Arg


        405                 410                 415


 


GAT GGT GGA AGT AAC ACC GGT GAC AAC AGG ACT GAG ACC
1285


Asp Gly Gly Ser Asn Thr Gly Asp Asn Arg Thr Glu Thr


                420                 425


 


TTT AGA CCT GGA GGA GGA GAT ATG AGG GAC AAT TGG AGA
1324


Phe Arg Pro Gly Gly Gly Asp Met Arg Asp Asn Trp Arg


    430                 435                 440


 


AGT GAA TTA TAT AAA TAT AAA GTA GTA AGA ATT GAA CCA
1363


Ser Glu Leu Tyr Lys Tyr Lys Val Val Arg Ile Glu Pro


            445                 450


 


TTA GGA GTA GCA CCC ACC CAG GCA AAG AGA AGA GTG GTG
1402


Leu Gly Val Ala Pro Thr Gln Ala Lys Arg Arg Val Val


455                 460                 465


 


CAA AGA GAA AAA AGA GCA GTG GGG ATA GGA GCT ATG TTC
1441


Gln Arg Glu Lys Arg Ala Val Gly Ile Gly Ala Met Phe


        470                 475                 480


 


CTT GGG TTC TTG GGA GAT AA 1461


Leu Gly Phe Leu Gly Asp


                 485 486


 








CLONE C8.6










G   GTA CCT GTG TGG AAA GAA GCA ACC ACC ACT CTA TTT
37



    Val Pro Val Trp Lys Glu Ala Thr Thr Thr Leu Phe


      1               5                  10


 


TGT GCA TCA GAT GCT AAA GCA TAT GAT ACA GAG GTA CAT
76


Cys Ala Ser Asp Ala Lys Ala Tyr Asp Thr Glu Val His


         15                  20                  25


 


AAT GTT TGG GCT ACA CAT GCC TGT GTA CCC ACA GAC CCC
115


Asn Val Trp Ala Thr His Ala Cys Val Pro Thr Asp Pro


                30                  35


 


AAC CCA CAA GAA GTA GTA TTG GAA AAT GTA ACA GAA AAT
154


Asn Pro Gln Glu Val Val Leu Glu Asn Val Thr Glu Asn


     40                  45                  50


 


TTT AAC ATG TGG AAA AAT AAC ATG GTA GAA CAG ATG CAT
193


Phe Asn Met Trp Lys Asn Asn Met Val Glu Gln Met His


             55                  60


 


GAG GAT ATA ATC AGT TTA TGG GAT CAA AGT CTA AAG CCA
232


Glu Asp Ile Ile Ser Leu Trp Asp Gln Ser Leu Lys Pro


 65                  70                  75


 


TGT GTA AAA TTA ACC CCA CTC TGT GTT ACT TTA AAT TGC
271


Cys Val Lys Leu Thr Pro Leu Cys Val Thr Leu Asn Cys


         80                  85                  90


 


ACT AAT TTG GAG AAT GCT AAT AAT ACC GAG AAT GCT AAT
310


Thr Asn Leu Glu Asn Ala Asn Asn Thr Glu Asn Ala Asn


                 95                 100


 


AAT ACC AAT AAT TAT ACC TTG GGG ATG GAG AGA GGT GAA
349


Asn Thr Asn Asn Tyr Thr Leu Gly Met Glu Arg Gly Glu


    105                 110                 115


 


AGA AAA AAC TGC TCT TTC AAT ATC ACC ACA AGC TTA AGA
388


Arg Lys Asn Cys Ser Phe Asn Ile Thr Thr Ser Leu Arg


            120                 125


 


GAT AAG GGG AAA AAA GAA TAT GCA TTG TTT TAT AAA CTT
427


Asp Lys Gly Lys Lys Glu Tyr Ala Leu Phe Tyr Lys Leu


130                 135                 140


 


GAT GTA GTA CAA ATA GAT AAT AGT ACC AAC TAT AGG CTG
466


Asp Val Val Gln Ile Asp Asn Ser Thr Asn Tyr Arg Leu


        145                 150                 155


 


ATA AGT TGT AAT ACC TCA GTC ATT ACA CAG GCC TGT CCA
505


Ile Ser Cys Asn Thr Ser Val Ile Thr Gln Ala Cys Pro


                160                 165


 


AAG GTA TCC TTT GAG CCA ATT CCC ATA CAT TAT TGT GCC
544


Lys Val Ser Phe Glu Pro Ile Pro Ile His Tyr Cys Ala


    170                 175                 180


 


CCG GCT GGT TTT GCG ATT CTA AAG TGT AAA GAT AAG AAG
583


Pro Ala Gly Phe Ala Ile Leu Lys Cys Lys Asp Lys Lys


            185                 190


 


TTC AAT GGA ACA GGA CCA TGT AAA AAT GTC AGG ACA GTA
622


Phe Asn Gly Thr Gly Pro Cys Lys Asn Val Arg Thr Val


195                 200                 205


 


CAA TGT ACA CAT GGA ATT AGA CCA GTA GTA TCA ACT CAA
661


Gln Cys Thr His Gly Ile Arg Pro Val Val Ser Thr Gln


         210                215                 220


 


CTA CTG TTA AAT GGC AGT CTA GCA GAA GAA GAG ATA GTA
700


Leu Leu Leu Asn Gly Ser Leu Ala Glu Glu Glu Ile Val


                225                 230


 


ATT AGA TCT GAA AAT ATC ACA GAC AAT GCT AAA ACC ATA
739


Ile Arg Ser Glu Asn Ile Thr Asp Asn Ala Lys Thr Ile


    235                 240                 245


 


ATA GTG GAG CTA AAT GAA TCT ATA GTG ATT AAT TGT ACA
778


Ile Val Gln Leu Asn Glu Ser Ile Val Ile Asn Cys Thr


            250                 255


 


AGA CCC AAT AAC AAC ACA AGA AAA AGT ATA AAT ATA GGA
817


Arg Pro Asn Asn Asn Thr Arg Lys Ser Ile Asn Ile Gly


260                 265                 270


 


CCA GGG AGA GCA TTC TAT ACA ACA GGA GAC ATA ATA GGA
856


Pro Gly Arg Ala Phe Tyr Thr Thr Gly Asp Ile Ile Gly


        275                 280                 285


 


GAT ATA AGA CAA GCA CAT TGT AAC CTT AGT AAA ACA CAA
895


Asp Ile Arg Gln Ala His Cys Asn Leu Ser Lys Thr Gln


                290                 295


 


TGG GAA AAA ACG TTA AGA CAG ATA GCT ATA AAA TTA GAA
934


Trp Glu Lys Thr Leu Arg Gln Ile Ala Ile Lys Leu Glu


    300                 305                 310


 


GAA AAA TTT AAG AAT AAA ACA ATA GCC TTT AAT AAA TCC
973


Glu Lys Phe Lys Asn Lys Thr Ile Ala Phe Asn Lys Ser


            315                 320


 


TCA GGA GGG GAC CCA GAA ATT GTA ATG CAC AGT TTT AAT
1012


Ser Gly Gly Asp Pro Glu Ile Val Met His Ser Phe Asn


325                 330                 335


 


TGT GGA GGG GGA TTT TTC TAC TGT AGT ACG AGA AAA CTG
1051


Cys Gly Gly Gly Phe Phe Tyr Cys Ser Thr Arg Lys Leu


        340                 345                 350


 


TTT AAT AGT ACC TGG AAT TTA ACA CAA CCG TTT AGT AAT
1090


Phe Asn Ser Thr Trp Asn Leu Thr Gln Pro Phe Ser Asn


                355                 360


 


ACC GGG GAT CGT ACT GAA GAG TTA AAT ATT ACA CTC CCA
1129


Thr Gly Asp Arg Thr Glu Glu Leu Asn Ile Thr Leu Pro


    365                 370                 375


 


TGC AGA ATA AAA CAA ATC ATA AAC TTG TGG CAG GAA GTA
1168


Cys Arg Ile Lys Gln Ile Ile Asn Leu Trp Gln Glu Val


            380                 385


 


GGC AAA GCA ATG TAT GCC CCT CCC ATC AGA GGA CAA ATT
1207


Gly Lys Ala Met Tyr Ala Pro Pro Ile Arg Gly Gln Ile


390                 395                 400


 


AGA TGT TCA TCA AAT ATT ACA GGG CTA CTA TTA AGG AGA
1246


Arg Cys Ser Ser Asn Ile Thr Gly Leu Leu Leu Arg Arg


        405                 410                 415


 


GAT GGT GGA AGT AAC ACC AGT GAC AAC CAG ACT GAG ACC
1285


Asp Gly Gly Ser Asn Thr Ser Asp Asn Gln Thr Glu Thr


                420                 425


 


TTT AGA CCT GGG GGA GGA GAT ATG AGG GAC AAG TGG AGA
1324


Phe Arg Pro Gly Gly Gly Asp Met Arg Asp Lys Trp Arg


    430                 435                 440


 


AGT GAA TTA TAT AAA TAT AAA GTA GTA AGA ATT GAA CCA
1363


Ser Glu Leu Tyr Lys Tyr Lys Val Val Arg Ile Glu Pro


            445                 450


 


TTA GGA GTA GCA CCC ACC CAG GCA AAG AGA AGA GTG GTG
1402


Leu Gly Val Ala Pro Thr Gln Ala Lys Arg Arg Val Val


455                 460                 465


 


CAA AGA GAA AAA AGA GCA GTG GGG ATA GGA GCT ATG TTC
1441


Gln Arg Glu Lys Arg Ala Val Gly Ile Gly Ala Met Phe


        470                 475                 480


 


CTT AGG TTC TTA GGA GAT AAA GCT TCT AGA GTC 1474


Leu Arg Phe Leu Gly Asp Lys Ala Ser Arg Val


                485                 490 491


 








CLONE C15.2










    CTC GAG GTA CCT GTA TGG AAA GAA GCA ACT ACC ACT
36



    Leu Glu Val Pro Val Trp Lys Glu Ala Thr Thr Thr


      1               5                  10


 


CTA TTT TGT GCA TCA GAT GCT AAA GCA TAT AAT ACA GAG
75


Leu Phe Cys Ala Ser Asp Ala Lys Ala Tyr Asn Thr Glu


         15                  20                  25


 


AAA CAT AAT GTT TGG GCC ACA CAC GCC TGT GTA CCC ACA
114


Lys His Asn Val Trp Ala Thr His Ala Cys Val Pro Thr


                 30                  35


 


GAT CCC AAC CCA CAA GAA GTA GTA TTG GGA AAT GTG ACA
153


Asp Pro Asn Pro Gln Glu Val Val Leu Gly Asn Val Thr


     40                  45                  50


 


GAA AAT TTT AAC ATG TGG AAA AAT AAC ATG GTA GAA CAA
192


Glu Asn Phe Asn Met Trp Lys Asn Asn Met Val Glu Gln


             55                  60


 


ATG CAT GAA GAT ATA ATC AGT TTA TGG GAT CAA AGT CTA
231


Met His Glu Asp Ile Ile Ser Leu Trp Asp Gln Ser Leu


 65                  70                  75


 


AAG CCA TGT GTA AAA TTA ACC CCA CTC TGT GTT ACT TTA
270


Lys Pro Cys Val Lys Leu Thr Pro Leu Cys Val Thr Leu


         80                  85                  90


 


AAT TGC ACT GAT GAT TTA GGG AAT GCT ACT AAT ACC AAT
309


Asn Cys Thr Asp Asp Leu Gly Asn Ala Thr Asn Thr Asn


                 95                 100


 


AGT AGT GCC ACT ACC AAT AGT AGT AGT TGG GAA GAA ATG
348


Ser Ser Ala Thr Thr Asn Ser Ser Ser Trp Glu Glu Met


    105                 110                 115


 


AAG GGG GAA ATG AAA AGA TGC TCT TTC AAT ATC ACC ACA
387


Lys Gly Glu Met Lys Arg Cys Ser Phe Asn Ile Thr Thr


            120                 125


 


AGC ATA AGA GAT AAG ATT AAG AAA GAA CAT GCA CTT TTC
426


Ser Ile Arg Asp Lys Ile Lys Lys Glu His Ala Leu Phe


130                 135                 140


 


TAT AGA CTT GAT GTA GTA CCA ATA GAT AAT GAT AAT ACC
465


Tyr Arg Leu Asp Val Val Pro Ile Asp Asn Asp Asn Thr


         145                  150                155


 


ACA TAT AGG TTG ATA AAT TGT AAT ACC TCA GTC ATT ACA
504


Thr Tyr Arg Leu Ile Asn Cys Asn Thr Ser Val Ile Thr


                160                 165


 


GAG GCC TGT CGA AAG GTA TCA TTT GAG CCA ATT CCC ATA
543


Gln Ala Cys Pro Lys Val Ser Phe Glu Pro Ile Pro Ile


    170                 175                 180


 


CAT TTT TGT GCC CGG GCT GGT TTT GCG ATT CTA AAG TGT
582


His Phe Cys Ala Pro Ala Gly Phe Ala Ile Leu Lys Cys


            185                 190


 


AAT AAT AAG ACG TTC GAG GGA AAA GGA CCA TGT AAA AAT
621


Asn Asn Lys Thr Phe Glu Gly Lys Gly Pro Cys Lys Asn


195                 200                 205


 


GTC AGT ACA GTA CAA TGC ACA CAT GGA ATT AGG CCA GTA
660


Val Ser Thr Val Gln Cys Thr His Gly Ile Arg Pro Val


        210                 215                 220


 


GTG TGA ACT GAA CTG CTG TTA AAT GGC AGT CTA GCA GAA
699


Val Ser Thr Gln Leu Leu Leu Asn Gly Ser Leu Ala Glu


                225                 230


 


GAA GAG GTA ATA ATT AGA TCT GAG AAT ATC ACA GAG AAT
738


Glu Glu Val Ile Ile Arg Ser Asp Asn Ile Thr Asp Asn


    235                 240                 245


 


ACT AAA AGG ATT ATA GTA GAG GTA AAG GAA TGT GTA GTA
777


Thr Lys Thr Ile Ile Val Gln Leu Asn Glu Ser Val Val


            250                 255


 


ATT AAT TGT AGA AGA CCC AAC AAC AAT ACA AGA AAA AGT
816


Ile Asn Cys Thr Arg Pro Asn Asn Asn Thr Arg Lys Ser


260                 265                 270


 


ATA CAT ATA GGA CGA GGG AGT GGA TTT TTT GCA ACA GGA
855


Ile His Ile Gly Pro Gly Ser Ala Phe Phe Ala Thr Gly


        275                 280                 285


 


GAA ATA ATA GGA GAT ATA AGA CAA GCA GAG TGT AAC CTT
894


Glu Ile Ile Gly Asp Ile Arg Gln Ala His Cys Asn Leu


                290                 295


 


AGT AGA ACA CAA TGG AAT AAC ACT TTA GGA AAG ATA GTG
933


Ser Arg Thr Gln Trp Asn Asn Thr Leu Gly Lys Ile Val


    300                 305                 310


 


ATA AAA TTA AGA GAA CAA TTT AGA AAA GAA TTT GGA GAA
972


Ile Lys Leu Arg Glu Gln Phe Arg Lys Gln Phe Gly Glu


            315                 320


 


AAA ACA ATA GTC TTT AAT CGA TCC TGA GGA GGG GAG CCG
1011


Lys Thr Ile Val Phe Asn Arg Ser Ser Gly Gly Asp Pro


325                 330                 335


 


GAA ATT GGA ATG GAG AGT TTT AAT TGT GGA GGG GAA TTT
1050


Glu Ile Ala Met His Ser Phe Asn Cys Gly Gly Glu Phe


        340                 345                 350


 


TTC TAG TGT AAG AGA ACA GGA GTG TTT AAT AGT AGG TGG
1089


Phe Tyr Gys Asn Thr Thr Ala Leu Phe Asn Ser Thr Trp


                355                 360


 


AAT GTT ACT AAA GGG TTG AAT AAC AGT GAA GGA AAT AGG
1128


Asn Val Thr Lys Gly Leu Asn Asn Thr Glu Gly Asn Ser


    365                 370                 375


 


ACA GGA GAT GAA AAT ATC ATA CTC GGA TGT AGA ATA AAA
1167


Thr Gly Asp Glu Asn Ile Ile Leu Pro Gys Arg Ile Lys


            380                 385


 


CAA ATT ATA AAG ATG TGG GAG GAA GTA GGA AAA GGA ATG
1206


Gln Ile Ile Asn Met Trp Gln Glu Val Gly Lys Ala Met


390                 395                 400


 


TAT GGG GGT CCC ATC AGT GGA GAA ATT AGA TGT TGA TGA
1245


Tyr Ala Pro Pro Ile Ser Gly Gln Ile Arg Gys Ser Ser


        405                 410                 415


 


AAG ATT AGA GGG GTG GTA CTA ACA AGA GAT GGT GGT AGT
1284


Asn Ile Thr Gly Leu Leu Leu Thr Arg Asp Gly Gly Ser


                420                 425


 


AAG AAC GAG AGC ATC ACC ACC GAG GTC TTC AGA CCT GGA
1323


Lys Asn Glu Ser Ile Thr Thr Glu Val Phe Arg Pro Gly


    430                 435                 440


 


GGA GGA GAT ATG AGG GAC AAT TGG AGA AGT GAA TTA TAT
1362


Gly Gly Asp Met Arg Asp Asn Trp Arg Ser Glu Leu Tyr


            445                 450


 


AAA TAT AAA GTA GTA AAA ATT GAA CCA TTA GGA GTA GCG
1401


Lys Tyr Lys Val Val Lys Ile Glu Pro Leu Gly Val Ala


455                 460                 465


 


CCC ACC AAG GCA AAG AGA AGA GTG GTG CAG AGA GAA AAA
1440


Pro Thr Lys Ala Lys Arg Arg Val Val Gln Arg Glu Lys


        470                 475                 480


 


AGA GCA GTG GGA ACA ATA GGA GCT ATG TTC CTT GGG TTC
1479


Arg Ala Val Gly Thr Ile Gly Ala Met Phe Leu Gly Phe


                485                 490


 


TTG GGA GCA TAA AGC TTC TAG ACT CGA CCT GCA 1512


Leu Gly Ala Xaa Ser Phe Xaa Ser Arg Pro Ala


    495                 500             504


 








CLONE C15.3










    CTC GAG GTA CCT GTC TGG AAA GAA GCA ACT ACC ACT
36



    Leu Glu Val Pro Val Trp Lys Glu Ala Thr Thr Thr


      1               5                  10


 


CTA TTT TGT GCA TCA CAT GCT AAA GCA TAT AAT ACA GAG
75


Leu Phe Cys Ala Ser Asp Ala Lys Ala Tyr Asn Thr Glu


         15                  20                  25


 


AAA CAT AAT GTT TGG GCC ACA CAC GCC TCT GTA CCC ACA
114


Lys His Asn Val Trp Ala Thr His Ala Cys Val Pro Thr


                 30                  35


 


GAT CCC AAC CCA CAA GAA CTA GTA TTG GGA AAT GTG ACA
153


Asp Pro Asn Pro Gln Glu Val Val Leu Gly Asn Val Thr


     40                  45                  50


 


GAA AAT TTT AAC ATG TCG AAA AAT AAC ATG GTA GAA CAA
192


Glu Asn Phe Asn Met Trp Lys Asn Asn Met Val Glu Gln


             55                  60


 


ATG CAT GAA GAT ATA ATC AGT TTA TGG CAT CAA AGT CTA
231


 Met His Glu Asp Ile Ile Ser Leu Trp Asp Gln Ser Leu


  65                  70                  75


 


AAG CCA TGT CTA AAA TTA ACC CCA CTC TGT GTT ACT TTA
270


Lys Pro Cys Val Lys Leu Thr Pro Leu Cys Val Thr Leu


         80                  85                  90


 


AAT TGC ACT GAT GAT TTA CCC AAT GCT ACT AAT ACC AAT
309


Asn Cys Thr Asp Asp Leu Gly Asn Ala Thr Asn Thr Asn


                 95                   100


 


AGC ACT GCC ACT ACC AAT AGT ACT AGT TGC GAA GAA ATG
348


Ser Ser Ala Thr Thr Asn Ser Ser Ser Trp Glu Glu Met


    105                 110                     115


 


AAG GGG GAA ATG AAA AGG TGC TCT TTC AAT ATC ACC ACA
387


Lys Gly Glu Met Lys Arg Cys Ser Phe Asn Ile Thr Thr


            120                 125


 


AGC ATA AGA CAT AAC ATT AAG AAA CAA CAT GCA CTT TTC
426


Ser Ile Arg Asp Lys Ile Lys Lys Glu His Ala Leu Phe


130                 135                 140


 


TAT AGA CTT GAT CTA GTA CCA ATA CAT AAT CAT AAT ACC
465


Tyr Arg Leu Asp Val Val Pro Ile Asp Asn Asp Asn Thr


        145                 150                 155


 


ACA TAT AGG TTG ATA AAT TGT AAT ACC TCA GTC ATT ACA
504


Thr Tyr Arg Leu Ile Asn Cys Asn Thr Ser Val Ile Thr


                160                 165


 


CAG CCC TGT CCA AAG GTA TCA TTT GAG CCA ATT CCC ATA
543


Gln Ala Cys Pro Lys Val Ser Phe Clu Pro Ile Pro Ile


    170                 175                 180


 


CAT TTT TCT CCC CCC CCT CCT TTT CCC ATT CTA AAG TCT
582


His Phe Cys Ala Pro Ala Gly Phe Ala Ile Leu Lys Cys


            185                 190


 


AAT AAT AAG ACG TTC GAG GGA AAA GGA CCA TGT AAA AAT
621


Asn Asn Lys Thr Phe Glu Gly Lys Gly Pro Cys Lys Asn


195                 200                 205


 


GTC AGT ACA GTA CAA TGC ACA CAT GGA ATT AGG CCA GTA
660


Val Ser Thr Val Gln Cys Thr His Gly Ile Arg Pro Val


        210                 215                 220


 


GTG TCA ACT CAA CTG CTG TTA AAT GGC AGT CTA GCA GAA
699


Val Ser Thr Gln Leu Leu Leu Asn Gly Ser Leu Ala Glu


                225                 230


 


GAA GAG GTA ATA ATT AGA TCT GGC AAT ATC ACA GAC AAT
738


Glu Glu Val Ile Ile Arg Ser Gly Asn Ile Thr Asp Asn


    235                 240                 245


 


ACT AAA ACC ATT ATA GTA CAG CTA AAC GAA TCT GTA GTA
777


Thr Lys Thr Ile Ile Val Gln Leu Asn Glu Ser Val Val


            250                 255


 


ATT AAT TGT ACA AGA TCC AAC AAC AAT ACA AGA AAA AGT
816


Ile Asn Cys Thr Arg Ser Asn Asn Asn Thr Arg Lys Ser


260                 265                 270


 


ATA CAT ATA GGA CCA GGG AGT GCA TTT TTT GCA ACA GGA
855


Ile His Ile Gly Pro Gly Ser Ala Phe Phe Ala Thr Gly


        275                 280                 285


 


GAA ATA ATA GGA GAT ATA AGA CAA GCA CAC TGT AAC CTT
894


Glu Ile Ile Gly Asp Ile Arg Gln Ala His Cys Asn Leu


                290                 295


 


AGT AGA ACA CAA TGG AAT AAC ACT TTA GGA AAG ATA GTC
933


Ser Arg Thr Gln Trp Asn Asn Thr Leu Gly Lys Ile Val


    300                 305                 310


 


ATA AAA TTA AGA GAA CAA TTT AGA AAA CAA TTT GGA GAA
972


Ile Lys Leu Arg Glu Gln Phe Arg Lys Gln Phe Gly Glu


            315                 320


 


AAA ACA ATA GTC TTT AAT CGA TCC TCA GGA GGG GAC CCG
1011


Lys Thr Ile Val Phe Asn Arg Ser Ser Gly Gly Asp Pro


325                 330                 335


 


GAA ATT GCA ATG CAC AGT TTT AAT TGT GGA GGG GAA TTT
1050


Glu Ile Ala Met His Ser Phe Asn Cys Gly Gly Glu Phe


        340                 345                 350


 


TTC TAC TGT AAC ACA ACA GCA CTG TTT AAT AGT ACC TGG
1089


Phe Tyr Cys Asn Thr Thr Ala Leu Phe Asn Ser Thr Trp


                355                 360


 


AAT GTT ACT AAA COG TTG AAT AAC ACT GAA GGA AAT AGC
1128


Asn Val Thr Lys Gly Leu Asn Asn Thr Glu Gly Asn Ser


    365                 370                 375


 


ACA GGG GAT GAA AAT ATC ATA CTC CCA TGT AGA ATA AAA
1167


Thr Gly Asp Glu Asn Ile Ile Leu Pro Cys Arg Ile Lys


            380                 385


 


CAA ATT ATA AAC ATG TGG CAG GAA GTA GGA AAA GCA ATG
1206


Gln Ile Ile Asn Met Trp Gln Glu Val Gly Lys Ala Met


390                 395                 400


 


TAT GCC CCT CCC ATC AGT GGA CAA ATT AGA TGT TCA TCA
1245


Tyr Ala Pro Pro Ile Ser Gly Gln Ile Arg Cys Ser Ser


        405                 410                 415


 


AAT ATT ACA COO CTG CTA CTA ACA AGA GAT GGT GGT AGT
1284


Asn Ile Thr Gly Leu Leu Leu Thr Arg Asp Gly Gly Ser


                420                 425


 


AAG AAC GAG ACC ATC ACC ACC GAG GTC TTC AGA CCT GGA
1323


Lys Asn Glu Ser Ile Thr Thr Glu Val Phe Arg Pro Gly


    430                 435                 440


 


GGA CGA GAT ATG AGG GAC AAT TGG AGA AGT GAA TTA TAT
1362


Gly Gly Asp Met Arg Asp Asn Trp Arg Ser Glu Leu Tyr


            445             450


 


AAA TAT AAA GTA GTA AAA ATT GAA CCA TTA GGA GTA GCG
1401


Lys Tyr Lys Val Val Lys Ile Glu Pro Leu Gly Val Ala


455                 460                 465


 


CCC ACC AAG GCA AAG AGA AGA GTG GTG CAG AGA GAA AAA
1440


Pro Thr Lys Ala Lys Arg Arg Val Val Gln Arg Glu Lys


        470                 475                 480


 


AGA GCA GTG GGA ACA ATA GGA GCT ATG TTC CTT GGG TTC
1479


Arg Ala Val Gly Thr Ile Gly Ala Met Phe Leu Gly Phe


                485                 490


 


TTA GGA GCA TAA AGC TTC TAG A 1501


Leu Gly Ala Xaa Ser Phe Xaa


    495                 500


 








CLONE C7.2










GG  GAA TTC GGA TCC GGG GTA CCT GTG TGG AAG GAA GCA
38



    Glu Phe Gly Ser Gly Val Pro Val Trp Lys Glu Ala


      1               5                  10


 


ACC ACC ACT CTA TTC TGT GCA TCA GAT GCT AGA GCA TAT
77


Thr Thr Thr Leu Phe Cys Ala Ser Asp Ala Arg Ala Tyr


         15                  20                  25


 


GAC ACA GAG GTA CAT AAT GTT TGG GCC ACA CAT GCC TGT
116


Asp Thr Glu Val His Asn Val Trp Ala Thr His Ala Cys


                 30                 35


 


GTA CCC ACA GAC CCT AGT CCA CAA GAA GTA GTT TTG GAA
155


Val Pro Thr Asp Pro Ser Pro Gln Glu Val Val Leu Glu


     40                  45                  50


 


AAT GTG ACA GAA AAT TTT AAC ATG TGG AAA AAT AAC ATG
194


Asn Val Thr Glu Asn Phe Asn Met Trp Lys Asn Asn Met


             55                  60


 


GTA GAA CAA ATG CAT GAG GAT ATA ATT AGT TTA TGG GAT
233


Val Glu Gln Met His Glu Asp Ile Ile Ser Leu Trp Asp


 65                  70                  75


 


CAA AGC TTA AAG CCA TGT GTA AAA TTA ACC CCA CTC TGT
272


Gln Ser Leu Lys Pro Cys Val Lys Leu Thr Pro Leu Cys


         80                  85                  90


 


GTT ACT TTA AAT TGC AGT GAT TAT AGG AAT GCT ACT GAT
311


Val Thr Leu Asn Cys Ser Asp Tyr Arg Asn Ala Thr Asp


                 95                 100


 


TAT AAG AAT GCT ACT GAT ACC ACT AGT AGT AAC GAG GGA
350


Tyr Lys Asn Ala Thr Asp Thr Thr Ser Ser Asn Glu Gly


    105                 110                 115


 


AAG ATG GAG AGA GGA GAA ATA AAA AAC TGC TCT TTC AAT
389


Lys Met Glu Arg Gly Glu Ile Lys Asn Cys Ser Phe Asn


            120                 125


 


ATT ACC ACA AGC ATA AAA AAT AAG ATG CAG AAA GAA TAT
428


Ile Thr Thr Ser Ile Lys Asn Lys Met Gln Lys Glu Tyr


130                 135                 140


 


GCA CTT TTC TAT AAA CTT GAT ATA GTA CCA ATA GAT AAT
467


Ala Leu Phe Tyr Lys Leu Asp Ile Val Pro Ile Asp Asn


        145                 150                 155


 


ACA AGC TAT ACA TTG ATA AGT TGT AAC ACC TCA GTC ATT
506


Thr Ser Tyr Thr Leu Ile Ser Cys Asn Thr Ser Val Ile


                160                      165


 


ACA CAG GCC TGT CCA AAG GTA TCC TTT GAA CCA ACT CCC
545


Thr Gln Ala Cys Pro Lys Val Ser Phe Glu Pro Thr Pro


    170                 175                 180


 


ATA CAT TAT TGT GCT CCG GCT GGT TTT GCG ATT CTA AAG
584


Ile His Tyr Cys Ala Pro Ala Gly Phe Ala Ile Leu Lys


            185                 190


 


TGT AAT GAT AAG AAG TTC AGT GGA AAA GGA GAA TGT AAA
623


Cys Asn Asp Lys Lys Phe Ser Gly Lys Gly Glu Cys Lys


195                 200                 205


 


AAT GTC AGC ACA GTA CAA TGT ACA CAT GGA ATT AGG CCA
662


Asn Val Ser Thr Val Gln Cys Thr His Gly Ile Arg Pro


        210                 215                 220


 


GTA GTA TCA ACT CAA CTG CTG TTA AAT GGC AGT CTA GCA
701


Val Val Ser Thr Gln Leu Leu Leu Asn Gly Ser Leu Ala


                225                 230


 


GAA GAA GAG GTG GTA ATT AGA TCT GAC AAT TTC ATA GAC
740


Glu Glu Glu Val Val Ile Arg Ser Asp Asn Phe Ile Asp


    235                 240                 245


 


AAT ACT AAA ACC ATA ATA GTA CAG CTG AAA GAA TCT GTA
779


Asn Thr Lys Thr Ile Ile Val Gln Leu Lys Glu Ser Val


            250                 255


 


GAA ATT AAT TGT ATA AGA CCC AAC AAT AAT ACA AGA AAA
818


Glu Ile Asn Cys Ile Arg Pro Asn Asn Asn Thr Arg Lys


260                 265                 270


 


GGT ATA CAT ATA GGA CCA GGG AGA GCA TGG TAT GCA ACA
857


Gly Ile His Ile Gly Pro Gly Arg Ala Trp Tyr Ala Thr


        275                 280                 285


 


GGA GAA ATA GTA GGA GAT ATA AGA AAG GCA TAT TGT AAC
896


Gly Glu Ile Val Gly Asp Ile Arg Lys Ala Tyr Cys Asn


                290                 295


 


ATT AGT AGA ACA AAA TGG AAT AAC ACT TTA ATA CAG ATA
935


Ile Ser Arg Thr Lys Trp Asn Asn Thr Leu Ile Gln Ile


    300                 305                 310


 


GCT AAC AAA TTA AAA GAA AAA TAT AAT ACA ACA ATA AGC
974


Ala Asn Lys Leu Lys Glu Lys Tyr Asn Thr Thr Ile Ser


            315                 320


 


TTT AAT CGA TCC TCA GGA GGG GAC CCA GAA ATT GTA ACG
1013


Phe Asn Arg Ser Ser Gly Gly Asp Pro Glu Ile Val Thr


325                 330                 335


 


CAT AGT TTT AAT TGT GGA GGG GAG TTT TTC TAC TGT GAT
1052


His Ser Phe Asn Cys Gly Gly Glu Phe Phe Tyr Cys Asp


        340                 345                 350


 


TCA ACA CAA CTG TTT AAT AGT ACT TGG AAT TTA AAT GGT
1091


Ser Thr Gln Leu Phe Asn Ser Thr Trp Asn Leu Asn Gly


                355                 360


 


ACT TGG AAT TTT ACT GCA GGG TCA AAT GAA ACT GAA GGC
1130


Thr Trp Asn Phe Thr Ala Gly Ser Asn Glu Thr Glu Gly


    365                 370                 375


 


AAT ATC ACA CTC CCA TGC AGA ATA AAA CAA ATT ATA AAC
1169


Asn Ile Thr Leu Pro Cys Arg Ile Lys Gln Ile Ile Asn


            380                 385


 


AGG TGG CAG GAA GTA GGG AAA GCA ATG TAT GCC CCT CCC
1208


Arg Trp Gln Glu Val Gly Lys Ala Met Tyr Ala Pro Pro


390                 395                 400


 


ATC AGT GGA CAA ATA AAA TGC TCA TCA AAC ATT ACA GGG
1247


Ile Ser Gly Gln Ile Lys Cys Ser Ser Asn Ile Thr Gly


        405                 410                 415


 


ATG ATA TTA ACA AGG GAT GGT GGT AAC GAG AAC AAT AAT
1286


Met Ile Leu Thr Arg Asp Gly Gly Asn Glu Asn Asn Asn


                420                 425


 


GAG AGC AGT ACT ACT GAG ACC TTC AGA CCG GGA GGA GGA
1325


Glu Ser Ser Thr Thr Glu Thr Phe Arg Pro Gly Gly Gly


    430                 435                 440


 


GAT ATG AGG AAC AAT TGG AGA AGT GAA TTA TAT AAA TAT
1364


Asp Met Arg Asn Asn Trp Arg Ser Glu Leu Tyr Lys Tyr


            445                 450


 


AAA GTA GTA AAA ATT GAA CCA TTA GGA GTA GCA CCC ACC
1403


Lys Val Val Lys Ile Glu Pro Leu Gly Val Ala Pro Thr


455                 460                 465


 


AAG GCA AAG AGA AGA GTG GTG CAG AGA GAA AAA AGA GCA
1442


Lys Ala Lys Arg Arg Val Val Gln Arg Glu Lys Arg Ala


        470                 475                 480


 


GTG GGA GCG CTA GGA GCT ATG TTC CTT GGG TTC TTA GGA
1481


Val Gly Ala Leu Gly Ala Met Phe Leu Gly Phe Leu Gly


                485                 490


 


GCA TAA AGC TTC TAG ACC GAC TCT AGA GGA TCC 1514


Ala Xaa Ser Phe Xaa Thr Asp Ser Arg Gly Ser


    495                 500             504


 








CLONE C7.10










G   GTA CCT GTG TGG AAG GAA GCA ACC ACC ACT CTA TTC
37



    Val Pro Val Trp Lys Glu Ala Thr Thr Thr Leu Phe


      1               5                 10


 


TGT GCA TCA GAT GCT AGA GCA TAT GAC ACA GAG GTA CAT
76


Cys Ala Ser Asp Ala Arg Ala Tyr Asp Thr Glu Val His


         15                  20                  25


 


AAT GTT TGG GCC ACA CAT GCC TGT GTA CCC ACA GAC CCT
115


Asn Val Trp Ala Thr His Ala Cys Val Pro Thr Asp Pro


                 30                  35


 


AGT CCA CAA GAA GTA TTT TTG GGA AAT GTG ACA GAA AAT
154


Ser Pro Gln Glu Val Phe Leu Gly Asn Val Thr Glu Asn


    40                   45                  50


 


TTT AAT ATG TGG AAA AAT AAC ATG GTA GAA CAA ATG TAT
193


Phe Asn Met Trp Lys Asn Asn Met Val Glu Gln Met Tyr


             55                  60


 


GAG GAT ATA ATT AGT TTA TGG GAT CAA AGC TTA AAG CCA
232


Glu Asp Ile Ile Ser Leu Trp Asp Gln Ser Leu Lys Pro


 65                  70                  75


 


TGT GTA AAA TTA ACC CCA CTC TGT GTT ACT TTA AAT TGC
271


Cys Val Lys Leu Thr Pro Leu Cys Val Thr Leu Asn Cys


         80                  85                  90


 


AGT GAT TAT AGG AAT GCT ACT GAT TAT AAG AAT GCT ACT
310


Ser Asp Tyr Arg Asn Ala Thr Asp Tyr Lys Asn Ala Thr


                 95                 100


 


GAT ACC ACT AGT AGT AAC GAG GGA AAG ATG GAG AGA GGA
349


Asp Thr Thr Ser Ser Asn Glu Gly Lys Met Glu Arg Gly


    105                 110                 115


 


GAA ATA AAA AAC TGC TCT TTC AAT ATC ACC ACA AGC ATA
388


Glu Ile Lys Asn Cys Ser Phe Asn Ile Thr Thr Ser Ile


            120                 125


 


AAA AAT AAG ATG CAG AAA GAA TAT GCA CTT TTC TAT AAA
427


Lys Asn Lys Met Gln Lys Glu Tyr Ala Leu Phe Tyr Lys


130                 135                 140


 


CTT AAT ATA GTA CCA ATA GAT AAT ACA AGC TAT ACA TTG
466


Leu Asn Ile Val Pro Ile Asp Asn Thr Ser Tyr Thr Leu


        145                 150                 155


 


ATA AGT TGT AAC ACC TCA GTC ATT ACA CAG GCC TGT CCA
505


Ile Ser Cys Asn Thr Ser Val Ile Thr Gln Ala Cys Pro


                160                 165


 


AAG GTA TCC TTT GAA CCA ATT CCC ATA CAT TAT TGT GCT
544


Lys Val Ser Phe Glu Pro Ile Pro Ile His Tyr Cys Ala


    170                 175                 180


 


CCG GCT GGT TTT GCG ATT CTA AAG TGT AAT GAT AAG AAG
583


Pro Ala Gly Phe Ala Ile Leu Lys Cys Asn Asp Lys Lys


            185                 190


 


TTC AGT GGA AAA GGA GAA TGT AAA AAT GTC AGC ACA GTA
622


Phe Ser Gly Lys Gly Glu Cys Lys Asn Val Ser Thr Val


195                 200                 205


 


CAA TGT ACA CAT GGA ATT AGG CCA GTA GTA TCA ACT CAA
661


Gln Cys Thr His Gly Ile Arg Pro Val Val Ser Thr Gln


        210                 215                 220


 


CTG CTG TTA AAT GGC AGT CTA GCA GAA GAA GAG GTG GTA
700


Leu Leu Leu Asn Gly Ser Leu Ala Glu Glu Glu Val Val


                225                 230


 


ATT AGA TCT GAC AAT TTC ACA GAC AAT ACT AAA ACC ATA
739


Ile Arg Ser Asp Asn Phe Thr Asp Asn Thr Lys Thr Ile


    235                 240                 245


 


ATA GTA CAG CTG AAA GAA TCT GTA GAA ATT AAT TGT ATA
778


Ile Val Gln Leu Lys Glu Ser Val Glu Ile Asn Cys Ile


            250                 255


 


AGA CCC AAC AAT AAT ACA AGA AAA GGT ATA CAT ATA GGA
817


Arg Pro Asn Asn Asn Thr Arg Lys Gly Ile His Ile Gly


260                 265                 270


 


CCA GGG AGA GCA TGG TAT GCA ACA GGA GAA ATA GTA GGA
856


Pro Gly Arg Ala Trp Tyr Ala Thr Gly Glu Ile Val Gly


        275                 280                 285


 


CAT ATA AGA CAG GCA TAT TGT AAC ATT AGT AGA ACA AAA
895


Asp Ile Arg Gln Ala Tyr Cys Asn Ile Ser Arg Thr Lys


                290                 295


 


TGG PAT AAC ACT TTA ATA CAG ATA GCT AAC AAA TTA AAA
934


Trp Asn Asn Thr Leu Ile Gln Ile Ala Asn Lys Leu Lys


    300                 305                 310


 


GAA AAA TAT AAT ACA ACA ATA AGC TTT PAT CGA TCC TCA
973


Glu Lys Tyr Asn Thr Thr Ile Ser Phe Asn Arg Ser Ser


            315                 320


 


GGA GGC GAC CCA CAA ATT GTA ACC CAT AGT TTT PAT TGT
1012


Gly Gly Asp Pro Clu Ile Val Thr His Ser Phe Asn Cys


325                 330                 335


 


GGA GGG GPA TTT TTC TAC TCT PAT TCA ACA CPA CTG TTT
1051


Gly Gly Clu Phe Phe Tyr Cys Asn Ser Thr Gln Leu Phe


        340                 345                 350


 


PAT AGT ACT TGG PAT TTA PAT COT ACT TGG PAT TTT ACT
1090


Asn Ser Thr Trp Asn Leu Asn Gly Thr Trp Asn Phe Thr


                355                 360


 


GCA GGG TCA PAT GAA ACT CPA CCC PAT ATC ACA CTC CCA
1129


Ala Gly Ser Asn Glu Thr Glu Gly Asn Ile Thr Leu Pro


    365                 370                 375


 


TGC AGA ATA AAA CPA ATT ATA PAC AGG TGG CAC GPA GTA
1168


Cys Arg Ile Lys Gln Ile Ile Asn Arg Trp Gln Glu Val


            380                 385


 


GGA AAA GCA ATG TAT CCC CCT CCC ATC AGT GGA CPA ATA
1207


Gly Lys Ala Met Tyr Ala Pro Pro Ile Ser Gly Gln Ile


390                 395                 400


 


AGA TGC TCA TCA PAC ATT ACA CCC ATG ATA TTA ACA AGG
1246


Arg Cys Ser Ser Asn Ile Thr Gly Met Ile Leu Thr Arg


        405                 410                 415


 


CAT GGT COT PAC GAG PAC PAT PAT GAG AGC ACT ACT ACT
1285


Asp Gly Gly Asn Glu Asn Asn Asn Glu Ser Ser Thr Thr


                420                 425


 


GAG ACC TTC ACA CCC GGA GGA GGA CAT ATG ACG PAC PAT
1324


Glu Thr Phe Arg Pro Gly Gly Gly Asp Met Arg Asn Asn


    430                 435                 440


 


TCG ACA ACT CPA TTA TAT PAA TAT PAA CTA GTA AAA ATT
1363


Trp Arg Ser Glu Leu Tyr Lys Tyr Lys Val Val Lys Ile


            445                 450


 


GAG CCA TTA GGA GTA GCA CCC ACC CAC TCT AGA GGA TCC
1402


Glu Pro Leu Gly Val Ala Pro Thr Asp Ser Arg Gly Ser


455                 460                 465


 


TCT ACA 1408


Ser Arg


    469


 








CLONE C11.5










    GAG GTA CCT GTG TCC PAA CPA GCA ACC ACT ACT CTA
36



    Glu Val Pro Val Trp Lys Glu Ala Thr Thr Thr Leu


      1               5                  10


 


TTT TGT GCA TCA CAT CCT AAA GCA TAT GAC ACA COG GTG
75


Phe Cys Ala Ser Asp Ala Lys Ala Tyr Asp Thr Gly Val


         15                  20                  25


 


CAT PAT GTT TGG GCC ACA CAT CCC TCT CTA CCC ACA GAC
114


His Asn Val Trp Ala Thr His Ala Cys Val Pro Thr Asp


                 30                  35


 


CCC PAC CCA CPA CPA ATA CPA TTG CTA PAT GTG ACA CPA
153


Pro Asn Pro Gln Glu Ile Glu Leu Val Asn Val Thr Glu


     40                  45                  50


 


CAT TTT AAC ATG TCC AAA PAT AAA ATC GTA GAC CAG ATG
192


Asp Phe Asn Met Trp Lys Asn Lys Met Val Asp Gln Met


             55                  60


 


CAT GAG GAT ATA ATC AGT TTA TGG GAT GAA AGC CTA AAG
231


His Glu Asp Ile Ile Ser Leu Trp Asp Glu Ser Leu Lys


 65                  70                  75


 


CCA TGT GTA AAG TTA ACC CCA CTT TGT GTT ACT CTA AAC
270


Pro Cys Val Lys Leu Thr Pro Leu Cys Val Thr Leu Asn


         80                  85                  90


 


TGC AGT GAT GTG AAC AAT TCC ACA AAT CCT AAT GAT ACT
309


Cys Ser Asp Val Asn Asn Ser Thr Asn Pro Asn Asp Thr


                 95                 100


 


AAT ACT AAT TCC ACT AAT ACT ACT TCC TCT ACT CCT ACG
348


Asn Thr Asn Ser Thr Asn Thr Thr Ser Ser Thr Pro Thr


    105                 110                 115


 


GCC ACT ACT AGT AGC GAG GAA AAG ATG GAG AAG GGA GAA
387


Ala Thr Thr Ser Ser Glu Glu Lys Met Glu Lys Gly Glu


            120                 125


 


ATA AAA AAC TGC TCT TTC AAT ATC ACC ACA CAC ATG AAA
426


Ile Lys Asn Cys Ser Phe Asn Ile Thr Thr His Met Lys


130                 135                 140


 


GAT AAG GCA CAG AAA GAA TAT GCA CTT TTT TAT AAA CTT
465


Asp Lys Ala Gln Lys Glu Tyr Ala Leu Phe Tyr Lys Leu


        145                 150                 155


 


GAT ATA GTA CCA ATA GAT GAT AAT AAT GCC AGC TAT AGG
504


Asp Ile Val Pro Ile Asp Asp Asn Asn Ala Ser Tyr Arg


                160                 165


 


TTG ATA AGT TGT AAT ACC TCA GAC ATT ACA CAG GCC TGT
543


Leu Ile Ser Cys Asn Thr Ser Asp Ile Thr Gln Ala Cys


    170                 175                 180


 


CCA AAG GTG ACC TTT GAG CCA ATT CCC ATA CAT TAT TGT
582


Pro Lys Val Thr Phe Glu Pro Ile Pro Ile His Tyr Cys


            185                 190


 


GCC CCG GCT GGT TTT GCG ATT CTA AAG TGT AAA GAT AAG
621


Ala Pro Ala Gly Phe Ala Ile Leu Lys Cys Lys Asp Lys


195                 200                 205


 


AAG TTC AAT GGA ACA GGA CCA TGT TCA AAG GTC AGC ACA
660


Lys Phe Asn Gly Thr Gly Pro Cys Ser Lys Val Ser Thr


        210                 215                 220


 


GTA CAA TGT ACA CAT GGA ATT AGG CCA GTA GTA TCA ACT
699


Val Gln Cys Thr His Gly Ile Arg Pro Val Val Ser Thr


                225                 230


 


CAA CTG TTG TTA AAT GGC AGT CTT GCA GAA GAA GAA GTA
738


Gln Leu Leu Leu Asn Gly Ser Leu Ala Glu Glu Glu Val


    235                 240                 245


 


GTA ATT AGA TCT GTC AAT TTC ACA GAC AAT GCT AAA ATC
777


Val Ile Arg Ser Val Asn Phe Thr Asp Asn Ala Lys Ile


            250                 255


 


ATA ATA GTA CAG CTG AAA GAA CCT GTA GCA ATT AAT TGT
816


Ile Ile Val Gln Leu Lys Glu Pro Val Ala Ile Asn Cys


260                 265                 270


 


ACA AGA CCC AAC AAC AAT ACA AGA AAA GGT ATA CAT CTA
855


Thr Arg Pro Asn Asn Asn Thr Arg Lys Gly Ile His Leu


        275                 280                 285


 


GGA CCA GGG AGC ACA TTT TAT ACA ACA GGA GAA ATA ATA
894


Gly Pro Gly Ser Thr Phe Tyr Thr Thr Gly Glu Ile Ile


                290                 295


 


GGA GAC ATA AGA AAA GCA TAT TGC AAG ATT AGT AAA GAA
933


Gly Asp Ile Arg Lys Ala Tyr Cys Lys Ile Ser Lys Glu


    300                 305                 310


 


AAA TGG AAT AAC ACT TTA AGA CAG GTA GTT AAA AAA TTA
972


Lys Trp Asn Asn Thr Leu Arg Gln Val Val Lys Lys Leu


            315                 320


 


AGA GAA CAA TTT GGG AAT AAA ACA ATA ATT TTT AAT CGA
1011


Arg Glu Gln Phe Gly Asn Lys Thr Ile Ile Phe Asn Arg


325                 330                 335


 


TCC TCA GGA GGG GAC CCA GAA ATT GTA ATG CAC AGT TTT
1050


Ser Ser Gly Gly Asp Pro Glu Ile Val Met His Ser Phe


        340                 345                 350


 


AAC TGT GGA GGG GAG TTT TTC TAC TGT AAT ACA ACA CAA
1089


Asn Cys Gly Gly Glu Phe Phe Tyr Cys Asn Thr Thr Gln


                355                 360


 


CTG TTT AAT AGT ACT TGG AAT AAT ACT GAA GGG ACA AAT
1128


Leu Phe Asn Ser Thr Trp Asn Asn Thr Glu Gly Thr Asn


    365                 370                 375


 


AGC ACT GAA GGA AAT AGC ACA ATC ACA CTC CCA TGC AGA
1167


Ser Thr Glu Gly Asn Ser Thr Ile Thr Leu Pro Cys Arg


            380                 385


 


ATA AAA CAA ATT ATA AAT ATG TGG CAG GAA GTA GGA AAA
1206


Ile Lys Gln Ile Ile Asn Met Trp Gln Glu Val Gly Lys


390                 395                 400


 


GCA ACG TAT GCC CCT CCC ATC AGA GGA CGA ATT AGA TGC
1245


Ala Thr Tyr Ala Pro Pro Ile Arg Gly Arg Ile Arg Cys


        405                 410                 415


 


ATA TCA AAT ATT ACA GGA CTG CTA TTA ACA AGA GAT GGT
1284


Ile Ser Asn Ile Thr Gly Leu Leu Leu Thr Arg Asp Gly


                420                 425


 


GGT AGG AAT GTC ACA AAC AAT ACC GAA ACC TTC AGA CCT
1323


Gly Arg Asn Val Thr Asn Asn Thr Glu Thr Phe Arg Pro


    430                 435                 440


 


GGA GGA GGA GAC ATG AGG GAC AAT TGG AGA AGT GAA TTA
1362


Gly Gly Gly Asp Met Arg Asp Asn Trp Arg Ser Glu Leu


            445                 450


 


TAT AAA TAT AAA GTA GTA AAA GTT GAA CCA TTA GGA ATA
1401


Tyr Lys Tyr Lys Val Val Lys Val Glu Pro Leu Gly Ile


455                 460                 465


 


GCA CCC ACC AAG GCA AAG AGA AGA GTG GTG CAC AGA GAC
1440


Ala Pro Thr Lys Ala Lys Arg Arg Val Val His Arg Asp


        470                 475                 480


 


AAA AGA GCA GCA CTA GGA GCC TTG TTC CTT GGG TTC TTA
1479


Lys Arg Ala Ala Leu Gly Ala Leu Phe Leu Gly Phe Leu


                485                 490


 


GGA GCA TAA AAG CTT CTA GA 1499


Gly Ala Xaa Lys Leu Leu


     495             499


 








CLONE C11.7










    GAG GTA CCT GTA TGG AAA GAA GCA ACC ACT ACT CTA
36



    Glu Val Pro Val Trp Lys Glu Ala Thr Thr Thr Leu


      1               5                  10


 


TTT TGT GCA TCA GAT GCT AAA GCA TAT GAC ACA GAG GTG
75


Phe Cys Ala Ser Asp Ala Lys Ala Tyr Asp Thr Glu Val


         15                  20                  25


 


CAT AAT GTT TGG GCC ACA CAT GCC TGT GTA CCC ACA GAC
114


His Asn Val Trp Ala Thr His Ala Cys Val Pro Thr Asp


                 30                  35


 


CCC AAC CCA CAA GAA ATA GAA TTG GTA AAT GTG ACA GAA
153


Pro Asn Pro Gln Glu Ile Glu Leu Val Asn Val Thr Glu


     40                  45                  50


 


GAT TTT AAC ATG TGG AAA AAT AAA ATG GTA GAC CAG ATG
192


Asp Phe Asn Met Trp Lys Asn Lys Met Val Asp Gln Met


             55                  60


 


CAT GAG GAT ATA ATC AGT TTA TGG GAT GAA AGC CTA AAG
231


His Glu Asp Ile Ile Ser Leu Trp Asp Glu Ser Leu Lys


 65                  70                  75


 


CCA TGT GTA AAG TTA ACC CCA CTT TGT GTT ACT CTA AAC
270


Pro Cys Val Lys Leu Thr Pro Leu Cys Val Thr Leu Asn


         80                  85                  90


 


TGC AGT GAT GTG AAC AAT TCC ACA AAT CCT AAT GAT ACT
309


Cys Ser Asp Val Asn Asn Ser Thr Asn Pro Asn Asp Thr


                 95                 100


 


AAT ACT AAT TCC ACT AAT ACT ACT TCC TCT ACT CCT ACG
348


Asn Thr Asn Ser Thr Asn Thr Thr Ser Ser Thr Pro Thr


    105                 110                 115


 


CCC ACT ACT AGT AGC GAG GAA AAG ATG GAG AAG GGA GAA
387


Ala Thr Thr Ser Ser Glu Glu Lys Met Glu Lys Gly Glu


            120                 125


 


ATA AAA AAC TGC TCT TTC AAT ATC ACC ACA CAC ATG AAA
426


Ile Lys Asn Cys Ser Phe Asn Ile Thr Thr His Met Lys


130                 135                 140


 


GAT AAG GTA CAG AAA GAA TAT GCA CTT TTT TAT AAA CTT
465


Asp Lys Val Gln Lys Glu Tyr Ala Leu Phe Tyr Lys Leu


        145                 150                 155


 


CAT ATA GTA CCA ATA GAT GAT AAT AAT ACC AGC TAT AGG
504


Asp Ile Val Pro Ile Asp Asp Asn Asn Thr Ser Tyr Arg


                160                 165


 


TTG ATA AGT TGT AAT ACC TCA GTC ATT ACA CAG GCC TGT
543


Leu Ile Ser Cys Asn Thr Ser Val Ile Thr Gln Ala Cys


    170                 175                 180


 


CCA ATG GTG ACC TTT GAG CCA ATT CCC ATA CAT TAT TGT
582


Pro Met Val Thr Phe Glu Pro Ile Pro Ile His Tyr Cys


            185                 190


 


GCC CCG GCT GGT TTT GCG ATT CTA AAG TGT AAA GAT AAG
621


Ala Pro Ala Gly Phe Ala Ile Leu Lys Cys Lys Asp Lys


195                 200                 205


 


AAC TTC AAT GGA ACA GGA CCA TGT TCA AAG GTC AGC ACA
660


Lys Phe Asn Gly Thr Gly Pro Cys Ser Lys Val Ser Thr


        210                 215                 220


 


GTA CAA TGT ACA CAT GGA ATT AGG CCA GTA GTA TCA ACT
699


Val Gln Cys Thr His Gly Ile Arg Pro Val Val Ser Thr


                225                 230


 


CAA CTG TTG TTA AAT GGC AGT CTT GCA GAA GAA GAA GTA
738


Gln Leu Leu Leu Asn Gly Ser Leu Ala Glu Glu Glu Val


    235                 240                 245


 


GTA ATT AGA TCT GTC AAT TTC ACA GAC AAT GCT AAA ATC
777


Val Ile Arg Ser Val Asn Phe Thr Asp Asn Ala Lys Ile


            250                 255


 


ATA ATA GTA CAG CTG AAA GAA CCT GTA GCA ATT AAT TGT
816


Ile Ile Val Gln Leu Lys Glu Pro Val Ala Ile Asn Cys


260                 265                 270


 


ACA AGA CCC AAC AAC AAT ACA AGA AAA GGT ATA CAT CTA
855


Thr Arg Pro Asn Asn Asn Thr Arg Lys Gly Ile His Leu


        275                 280                 285


 


GGA CCA GGG AGC ACA TTT TAT ACA ACA GGA GAA ATA ATA
894


Gly Pro Gly Ser Thr Phe Tyr Thr Thr Gly Glu Ile Ile


                290                 295


 


GGA GAC ATA AGA AAA GCA TAT TGC AAG ATT AGT AAA GAA
933


Gly Asp Ile Arg Lys Ala Tyr Cys Lys Ile Ser Lys Glu


    300                 305                 310


 


AAA TGG AAT AAC ACT TTA AGA CAG GTA GTT AAA AAA TTA
972


Lys Trp Asn Asn Thr Leu Arg Gln Val Val Lys Lys Leu


            315                 320


 


AGA GAA CAA TTT GGG AAT AAA ACA ATA ATT TTT AAT CGA
1011


Arg Glu Gln Phe Gly Asn Lys Thr Ile Ile Phe Asn Arg


325                 330                 335


 


TCC TCA GGA GGG GAC CCA GAA ATT GTA ATG CAC AGT TTT
1050


Ser Ser Gly Gly Asp Pro Glu Ile Val Met His Ser Phe


        340                 345                 350


 


AAC TGT GGA GGG GAG TTT TTC TAC TGT AAT ACA ACA CAA
1089


Asn Cys Gly Gly Glu Phe Phe Tyr Cys Asn Thr Thr Gln


                355                 360


 


CTG TTT AAT AGT ACT TGG AAT AAT ACT GAA GGG ACA AAT
1128


Leu Phe Asn Ser Thr Trp Asn Asn Thr Glu Gly Thr Asn


    365                 370                 375


 


AGC ACT GAA GGA AAT AGC ACA ATC ACA CTC CCA TGC AGA
1167


Ser Thr Glu Gly Asn Ser Thr Ile Thr Leu Pro Cys Arg


            380                 385


 


ATA AAA CAA ATT ATA AAT ATG TGG CAG GAA GTA GGA AAA
1206


Ile Lys Gln Ile Ile Asn Met Trp Gln Glu Val Gly Lys


390                 395                 400


 


GCA ACG TAT GCC CCT CCC ATC AGA GGA CGA ATT AGA TGC
1245


Ala Thr Tyr Ala Pro Pro Ile Arg Gly Arg Ile Arg Cys


        405                 410                 415


 


ATA TCA AAT ATT ACA GGA CTG CTA TTA ACA AGA GAT GGT
1284


Ile Ser Asn Ile Thr Gly Leu Leu Leu Thr Arg Asp Gly


                420                 425


 


GGT AGG AAT GTC ACA AAC AAT ACC GAN NCC TTC AGA CCT
1323


Gly Arg Asn Val Thr Asn Asn Thr Xaa Xaa Phe Arg Pro


    430                 435                 440


 


GGA GGA GGA GAC ATG AGG GAC AAT TGG AGA AGT GAA TTA
1362


Gly Gly Gly Asp Met Arg Asp Asn Trp Arg Ser Glu Leu


            445                 450


 


TAT AAA TAT AAA GTA GTA AAA GTT GAA CCA TTA GGA ATA
1401


Tyr Lys Tyr Lys Val Val Lys Val Glu Pro Leu Gly Ile


455                 460                 465


 


GCA CCC ACC AAG GCA AAG AGA AGA GTG GTG CAC AGA GAC
1440


Ala Pro Thr Lys Ala Lys Arg Arg Val Val His Arg Asp


        470                 475                 480


 


AAA AGA GCA GCA CTA GGA GCT TTG TTC CTT GGG TTC TTA
1479


Lys Arg Ala Ala Leu Gly Ala Leu Phe Leu Gly Phe Leu


                485                 490


 


GGA GCA TAA AAG CTT CTA GA 1499


Gly Ala Xaa Lys Leu Leu


     495             499


 








CLONE C10.5










G   GTA CCT GTG TGG AAA GAA GCA AAC ACA ACT CTA TTT
37



    Val Pro Val Trp Lys Glu Ala Asn Thr Thr Leu Phe


      1                 5                 10


 


TGT GCA TCA GAT GCT AAA GCA TAT GAT AGA GAA GTA CAT
76


Cys Ala Ser Asp Ala Lys Ala Tyr Asp Arg Glu Val His


          15                  20                  25


 


AAT GTT TGG GCA ACA CAT GCC TGT GTA CCC ACA GAC CCC
115


Asn Val Trp Ala Thr His Ala Cys Val Pro Thr Asp Pro


                 30                  35


 


AAC CCA CAA GAA ATA GTA TTG GGA AAT GTG ACA GAA AAT
154


Asn Pro Gln Glu Ile Val Leu Gly Asn Val Thr Glu Asn


     40                  45                  50


 


TTT AAC ATG TGG AAA AAT AAC ATG GTA GAA CAA ATG CAT
193


Phe Asn Met Trp Lys Asn Asn Met Val Glu Gln Met His


             55                  60


 


GAG GAT ATA ATC AAT TTA TGG GAT CAA AGC TTA AAG CCA
232


Glu Asp Ile Ile Asn Leu Trp Asp Gln Ser Leu Lys Pro


 65                  70                 75


 


TGT GTA AAG TTA ACT CCA CTC TGT GTT ACT TTA AAG TGC
271


Cys Val Lys Leu Thr Pro Leu Cys Val Thr Leu Lys Cys


         80                  85                  90


 


AAG GAT CTG GAG AGG AAT ACT ACC TAT AAT AGC ACT ATT
310


Lys Asp Leu Glu Arg Aen Thr Thr Tyr Asn Ser Thr Ile


                 95                 100


 


ACC AAT AAT AGT AGT TTG GAG GGA CTA AGA GAA CAA ATG
349


Thr Asn Aen Ser Ser Leu Glu Gly Leu Arg Glu Gln Met


    105                 110                 115


 


ACA AAC TGC TCT TTC AAC ATC ACC ACA AGT ATA AGA GAT
388


Thr Asn Cys Ser Phe Asn Ile Thr Thr Ser Ile Arg Asp


            120                 125


 


AAG GTG CAG AAA GAA TAT GCA CTT TTG TAT AAA CTT GAT
427


Lys Val Gln Lys Glu Tyr Ala Leu Leu Tyr Lys Leu Asp


130                 135                 140


 


GTA GTA CCA ATA GAA GAA GAT GAC AAT ACT AGC TAT AGA
466


Val Val Pro Ile Glu Glu Asp Asp Asn Thr Ser Tyr Arg


        145                 150                 155


 


TTG ATA AGT TGT AAC ACC TCA GTC ATT ACA CAG GCT TGT
505


Leu Ile Ser Cys Asn Thr Ser Val Ile Thr Gln Ala Cys


                160                 165


 


CCA AAG ACA TCC TTT GAG CCA ATT CCC ATA CAT TAT TGT
544


Pro Lys Thr Ser Phe Glu Pro Ile Pro Ile His Tyr Cys


    170                 175                 180


 


GCC CCG GCT GGT TTT GCG ATT CTA AAG TGT AAT GAT AAG
583


Ala Pro Ala Gly Phe Ala Ile Leu Lys Cys Asn Asp Lys


            185                 190


 


AAG TTC AAT GGA ACA GGA CCA TGT AAA AAT GTC AGC ACA
622


Lys Phe Asn Gly Thr Gly Pro Cys Lys Asn Val Ser Thr


195                 200                 205


 


GTA CAA TGT ACA CAT GGA ATT AGG CCA GTA GTA TCA ACT
661


Val Gln Cys Thr His Gly Ile Arg Pro Val Val Ser Thr


        210                 215                 220


 


CAA CTG TTG TTA AAT GGC AGT CTA GCA GAA GAA GAG GTA
700


Gln Leu Leu Leu Asn Gly Ser Leu Ala Glu Glu Glu Val


                225                230


 


GTA ATC AGA TCT GCC AAT TTC ACA GAC AAT GCT AAA ACC
739


Val Ile Arg Ser Ala Asn Phe Thr Asp Asn Ala Lys Thr


    235                 240                 245


 


ATA ATA GTA CAT CTA AAT GAA ACT GTA AAA ATT AAT TGT
778


Ile Ile Val His Leu Asn Glu Thr Val Lys Ile Asn Cys


            250                 255


 


ACA AGA CTT GGC AAC AAT ACA AGA AAA AGT ATA AAT ATA
817


Thr Arg Leu Gly Asn Asn Thr Arg Lys Ser Ile Asn Ile


260                 265                 270


 


GGA CCA GGG AGA GTA CTC TAT GCA ACA GGA GAA ATA ATA
856


Gly Pro Gly Arg Val Leu Tyr Ala Thr Gly Glu Ile Ile


        275                 280                 285


 


GGA GAC ATA AGA CAA GCA CAT TGT AAC ATT AGT AGA GCA
895


Gly Asp Ile Arg Gln Ala His Cys Asn Ile Ser Arg Ala


                290                 295


 


CAA TGG AAT AAG ACT TTA GAA AAG GTA GTT GAC AAA TTA
934


Gln Trp Asn Lys Thr Leu Glu Lys Val Val Asp Lys Leu


    300                 305                 310


 


AGA AAA CAA TTT GGG GAT AAT ACA ACA ATA GCT TTT AAT
973


Arg Lys Gln Phe Gly Asp Asn Thr Thr Ile Ala Phe Asn


            315                 320


 


CGA TCC TCA GGA GGG GAC CCA GAA ATT GTA ATG CAC ACT
1012


Arg Ser Ser Gly Gly Asp Pro Glu Ile Val Met His Thr


325                 330                 335


 


TTT AAT TGT GGA GGG GAA TTT TTC TAC TGT AAT ACA ACA
1051


Phe Asn Cys Gly Gly Glu Phe Phe Tyr Cys Asn Thr Thr


        340                 345                 350


 


CAA CTG TTT AAT AGT ACT TGG AAT AAT ACT TGG AAG GAT
1090


Gln Leu Phe Asn Ser Thr Trp Asn Asn Thr Trp Lys Asp


                355                 360


 


CCT AAC AGG AGT GAC AAT ATC ACA CTC CCA TGC AGA ATA
1129


Pro Asn Arg Ser Asp Asn Ile Thr Leu Pro Cys Arg Ile


    365                 370                 375


 


AAA CAA ATT ATA AAC ATG TGG CAG GAA GTA GGA AAA GCA
1168


Lys Gln Ile Ile Asn Met Trp Gln Glu Val Gly Lys Ala


            380                 385


 


ATG TAC GCC CCT CCC ATC AGA GGG GAA ATT AGA TGT TCA
1207


Met Tyr Ala Pro Pro Ile Arg Gly Glu Ile Arg Cys Ser


390                 395                 400


 


TCA AAT ATC ACA GGG CTG CTA CTA ACA AGA GAT GGT GGT
1246


Ser Asn Ile Thr Gly Leu Leu Leu Thr Arg Asp Gly Gly


        405                 410                 415


 


AAT GAC GAT GGT AAT GAC ACG ACC ACA AAC AGG ACC GAG
1285


Asn Asp Asp Gly Asn Asp Thr Thr Thr Asn Arg Thr Glu


                420                 425


 


ATC TTC AGA CCT GGA GGA GGA GAT ATG AGG GAC AAT TGG
1324


Ile Phe Arg Pro Gly Gly Gly Asp Met Arg Asp Asn Trp


    430                 435                 440


 


AGA AGT GAA TTA TAT AGA TAT AAA GTA GTA AAA ATT GAA
1363


Arg Ser Glu Leu Tyr Arg Tyr Lys Val Val Lys Ile Glu


            445                 450


 


CCA TTA GGA ATA GCA CCC ACC AGG GCA AAG AGA AGA GTG
1402


Pro Leu Gly Ile Ala Pro Thr Arg Ala Lys Arg Arg Val


455                 460                 465


 


GTG CAG AGA GAA AAA AGA GCA GTA GGA CTA GGA GCT TTG
1441


Val Gln Arg Glu Lys Arg Ala Val Gly Leu Gly Ala Leu


        470                 475                 480


 


TTC CTT GGG T TCTTAGGAG CATAAAGCTT CTAGA 1475


Phe Leu Gly


        483


 








CLONE C10.7










G   GTA CCT GTG TGG AAA GAA GCA AAC ACA ACT CTA TTT
37



    Val Pro Val Trp Lys Glu Ala Asn Thr Thr Leu Phe


      1               5                  10


 


TGT GCA TCA GAT GCT AAA GCA TAT GAT AGA GAA GTA CAT
76


Cys Ala Ser Asp Ala Lys Ala Tyr Asp Arg Glu Val His


         15                  20                  25


 


AAT GTT TGG GCA ACA CAT GCC TGT GTA CCC ACA GAC CCC
115


Asn Val Trp Ala Thr His Ala Cys Val Pro Thr Asp Pro


                 30                  35


 


AAC CCA CAA GAA ATA GTA TTG GGA AAT GTG ACA GAA AAT
154


Asn Pro Gln Glu Ile Val Leu Gly Asn Val Thr Glu Asn


     40                  45                  50


 


TTT AAC ATG TGG AAA AAT AAC ATG GTA GAA CAA ATG CAT
193


Phe Asn Met Trp Lys Asn Asn Met Val Glu Gln Met His


             55                  60


 


GAG GAT ATA ATC AAT TTA TGG GAT CAA AGC TTA AAG CCA
232


Glu Asp Ile Ile Asn Leu Trp Asp Gln Ser Leu Lys Pro


 65                  70                  75


 


TGT GTA AAG TTA ACT CCA CTC TGT GTT ACT TTA AAG TGC
271


Cys Val Lys lAu Thr Pro Leu Cys Val Thr Leu Lys Cys


         80                  85                  90


 


AAG GAT CTG GAG AGG AAT ACT ACC TAT AAT AGC ACT ATT
310


Lys Asp Leu Glu Arg Asn Thr Thr Tyr Asn Ser Thr Ile


                 95                 100


 


ACC AAT AAT AGT AGT TTG GAG GGA CTA AGA GAA CAA ATG
349


Thr Asn Asn Ser Ser Leu Glu Gly Leu Arg Glu Gln Met


    105                 110                 115


 


ACA AAC TGC TCT TTC AAC ATC ACC ACA AGT ATA AGA GAT
388


Thr Asn Cys Ser Phe Asn Ile Thr Thr Ser Ile Arg Asp


            120                 125


 


AAG GTG CAG AAA GAA TAT GCA CTT TTG TAT AAA CTT GAT
427


Lys Val Gln Lys Glu Tyr Ala Leu Leu Tyr Lys Leu Asp


130                 135                 140


 


GTA GTA CCA ATA GAA GAA GAT GAC AAT ACT AGC TAT AGA
466


Val Val Pro Ile Glu 0Th Asp Asp Asn Thr Ser Tyr Arg


        145                 150                 155


 


TTG ATA AGT TGT AAC ACC TCA GTC ATT ACA CAG GCT TGT
505


Leu Ile Ser Cys Asn Thr Ser Val Ile Thr Gln Ala Cys


                160                 165


 


CCA AAG ACA TCC TTT GAG CCA ATT CCC ATA CAT TAT TGT
544


Pro Lys Thr Ser Phe Glu Pro Ile Pro Ile His Tyr Cys


    170                 175                 180


 


GCC CCG GCT GGT TTT GCG ATT CTA AAG TGT AAT GAT AAG
583


Ala Pro Ala Gly Phe Ala Ile Leu Lys Cys Asn Asp Lys


            185                 190


 


AAG TTC AAT GGA ACA GGA CCA TGT AAA AAT GTC AGC ACA
622


Lys Phe Asn Gly Thr Gly Pro Cys Lys Asn Val Ser Thr


195                 200                 205


 


GTA CAA TGT ACA CAT GGA ATT AGG CCA GTA GTA TCA ACT
661


Val Gln Cys Thr His Gly Ile Arg Pro Val Val Ser Thr


        210                 215                 220


 


CAA CTG TTG TTA AAT GGC AGT CTA GCA GAA GAA GAG GTA
700


Gln Leu Leu Leu Asn Gly Ser Leu Ala Glu Glu Glu Val


                225                 230


 


GTA ATC AGA TCT GCC AAT TTC ACA GAC AAT GCT AAA ACC
739


Val Ile Arg Ser Ala Asn Phe Thr Asp Asn Ala Lys Thr


    235                 240                 245


 


ATA ATA GTA CAT CTA AAT GAA ACT GTA AAA ATT AAT TGT
778


Ile Ile Val His Leu Asn Glu Thr Val Lys Ile Asn Cys


            250                 255


 


ACA AGA CTT GGC AAC AAT ACA AGA AAA AGT ATA AAT ATA
817


Thr Arg Leu Gly Asn Asn Thr Arg Lys Ser Ile Asn Ile


260                 265                 270


 


GGA CCA GGG AGA GTA CTC TAT GCA ACA GGA GAA ATA ATA
856


Gly Pro Gly Arg Val Leu Tyr Ala Thr Gly Glu Ile Ile


        275                 280                 285


 


GGA GAC ATA AGA CAA GCA CAT TGT AAC ATT AGT AGA GCA
895


Gly Asp Ile Arg Gln Ala His Cys Asn Ile Ser Arg Ala


                290                 295


 


CAA TGG AAT AAG ACT TTA GAA AAG GTA GTT GAC AAG TTA
934


Gln Trp Asn Lys Thr Leu Glu Lys Val Val Asp Lys Leu


    300                 305                 310


 


AGA AAA CAA TTT GGG GAT AAT ACA ACA ATA GCT TTT AAT
973


Arg Lys Gln Phe Gly Asp Asn Thr Thr Ile Ala Phe Asn


            315                 320


 


CGA TCC TCA GGA GGG GAC CCA GAA ATT GTA ATG CAC ACT
1012


Arg Ser Ser Gly Gly Asp Pro Glu Ile Val Met His Thr


325                 330                 335


 


TTT AAT TGT GGA GGG GAA TTT TTC TAC TGT AAT ACA ACA
1051


Phe Asn Cys Gly Gly Glu Phe Phe Tyr Cys Asn Thr Thr


        340                 345                 350


 


CAA CTG TTT AAT AGT ACT TGG AAT AAT ACT TGG AAG GAT
1090


Gln Leu Phe Asn Ser Thr Trp Asn Asn Thr Trp Lys Asp


                355                 360


 


CCT AAC AGG AGT GAC AAT ATC ACA CTC CCA TGC AGA ATA
1129


Pro Asn Arg Ser Asp Asn Ile Thr Leu Pro Cys Arg Ile


    365                 370                 375


 


AAA CAA ATT ATA AAC ATG TGG CAG GAA GTA GGA AAA GCA
1168


Lys Gln Ile Ile Asn Met Trp Gln Glu Val Gly Lys Ala


            380                 385


 


ATG TAC GCC CCT CCC ATC AGA GGG GAA ATT AGA TGT TCA
1207


Met Tyr Ala Pro Pro Ile Arg Gly Glu Ile Arg Cys Ser


390                 395                 400


 


TCA AAT ATC ACA GGG CTG CTA CTA ACA AGA GAT GGT GGT
1246


Ser Asn Ile Thr Gly Leu Leu Leu Thr Arg Asp Gly Gly


        405                 410                 415


 


AAT GAC GAT GGT AAT GAC ACG ACC ACA AAC AGG ACC GAG
1285


Asn Asp Asp Gly Asn Asp Thr Thr Thr Asn Arg Thr Glu


                420                 425


 


ATC TTC AGA CCT GGA GGA GGA GAT ATG AGG GAC AAT TGG
1324


Ile Phe Arg Pro Gly Gly Gly Asp Met Arg Asp Asn Trp


    430                 435                 440


 


AGA AGT GAA TTA TAT AGA TAT AAA GTA GTA AAA ATT GAA
1363


Arg Ser Glu Leu Tyr Arg Tyr Lys Val Val Lys Ile Glu


            445                 450


 


CCA TTA GGA ATA GCA CCC ACC AGG GCA AAG AGA AGA GTG
1402


Pro Leu Gly Ile Ala Pro Thr Arg Ala Lys Arg Arg Val


455                 460                 465


 


GTG CAG AGA GAA AAA AGA GCA GTA GGA CTA GGA GCT TTG
1441


Val Gln Arg Glu Lys Arg Ala Val Gly Leu Gly Ala Leu


        470                 475                 480


 


TTC CTT GGG TTC TTG GGA GCA TAA AGC TTC TAG A 1475


Phe Leu Gly Phe Leu Gly Ala Xaa Ser Phe Xaa


                485                 490 491


 








CLONE C17.1










    CTC GAG GTA CCT GTG TGG AAA GAA GCA ACC ACC ACT
36



    Leu Glu Val Pro Val Trp Lys Glu Ala Thr Thr Thr


      1              5                  10


 


CTA TTT TGT GCA TCA GAT GCT AAA GCA TAT GAT TCA GAG
75


Leu Phe Cys Ala Ser Asp Ala Lys Ala Tyr Asp Ser Glu


         15                  20                  25


 


GCA CAT AAT GTT TGG GCC ACA CAT GCC TGT GTA CCC ACA
114


Ala His Asn Val Trp Ala Thr His Ala Cys Val Pro Thr


                 30                  35


 


GAC CCC AAC CCA CAA GAA GTA GAA TTG GAA AAT GTG ACA
153


Asp Pro Asn Pro Gln Glu Val Glu Leu Glu Asn Val Thr


     40                  45                  50


 


GAA AAT TTT AAC ATG TGG AAA AAT AAC ATG GTA GAA CAG
192


Glu Asn Phe Asn Met Trp Lys Asn Asn Met Val Glu Gln


             55                  60


 


ATG CAT GGO GAT ATA ATT AGT TTA TGG GAT CAA AGC CTA
231


Met His Gly Asp Ile Ile Ser Leu Trp Asp Gln Ser Leu


 65                  70                  75


 


AAG CCA TGT GTA AAA TTA ACC CCA CTC TGT GTT ACG TTA
270


Lys Pro Cys Val Lys Leu Thr Pro Leu Cys Val Thr Leu


         80                  85                  90


 


AAT TGC ACT GAC CCA AAT GTT ACT AAT AGC GAG AGA ACG
309


Asn Cys Thr Asp Pro Asn Val Thr Asn Ser Glu Arg Thr


                 95                 100


 


ATA GAG GGG GGA GAA ATA AAA AAT TGC TCT TTC AAT ATC
348


Ile Glu Gly Gly Glu Ile Lys Asn Cys Ser Phe Asn Ile


    105                 110                 115


 


ACC ACA AAC ATA AGA GAT AGG TTT CAG AAA GAA TAT GCA
387


Thr Thr Asn Ile Arg Asp Arg Phe Gln Lys Glu Tyr Ala


            120                 125


 


CTT TTT TAT AAA CTT GAT GTA ATA CCA TTA GGT AAT GAT
426


Leu Phe Tyr Lys Leu Asp Val Ile Pro Leu Gly Asn Asp


130                 135                 140


 


AAT ACT AGC TAT AGG TTG ATA AGT TGT AAC ACC TCA GTC
465


Asn Thr Ser Tyr Arg Leu Ile Ser Cys Asn Thr Ser Val


        145                 150                 155


 


ATT ACA CAG GCC TGT CCA AAG GTA TCC TTT GAG CCA ATT
504


Ile Thr Gln Ala Cys Pro Lys Val Ser Phe Glu Pro Ile


                160                 165


 


CCC ATA CAT TAT TGT GCC CCG GCT GGT TTT GCG ATT CTA
543


Pro Ile His Tyr Cys Ala Pro Ala Gly Phe Ala Ile Leu


    170                 175                 180


 


AAG TGT AAA GAT AAG AAG TTC AAT GGA ACA GGA CCA TGT
582


Lys Cys Lys Asp Lys Lys Phe Asn Gly Thr Gly Pro Cys


            185                 190


 


ACA AAT GTC AGC ACA GTA CAA TGT ACA CAT GGA ATT AAG
621


Thr Asn Val Ser Thr Val Gln Cys Thr His Gly Ile Lys


195                 200                 205


 


CCA GTA GTA TCA ACT CAA CTG TTG TTA AAT GGC AGT CTA
660


Pro Val Val Ser Thr Gln Leu Leu Leu Asn Gly Ser Leu


        210                 215                 220


 


GCA GAA GAA GAC ATA GTA ATT AGA TCC GCC AAT CTC ACA
699


Ala Glu Glu Asp Ile Val Ile Arg Ser Ala Asn Leu Thr


                225                 230


 


GAC AAT GCT AAA AAC ATA ATA GTA CAG CTG AAT GAA TCT
738


Asp Asn Ala Lys Asn Ile Ile Val Gln Leu Asn Glu Ser


    235                 240                 245


 


GTA ACA ATG AAT TGT ACA AGA CCC AAC AAC AAT ACA ATG
777


Val Thr Met Asn Cys Thr Arg Pro Asn Asn Asn Thr Met


            250                 255


 


AAA AGT ATA CAT ATA GGA CCA GGC AGA GCA TTT TAT GCA
816


Lys Ser Ile His Ile Gly Pro Gly Arg Ala Phe Tyr Ala


260                 265                 270


 


ACA GGA AAC ATA ATA GGA GAT ATA AGA CAA GCA CAT TGT
855


Thr Gly Asn Ile Ile Gly Asp Ile Arg Gln Ala His Cys


        275                 280                 285


 


AAC ATT AGT GGA ACA AAA TGG AAT GAC ACT TTG AAA AAG
894


Asn Ile Ser Gly Thr Lys Trp Asn Asp Thr Leu Lys Lys


                290                 295


 


ATA GCT ATA AAA TTA AGA GAA CAA TTT AAT AAG ACA ATA
933


Ile Ala Ile Lys Leu Arg Glu Gln Phe Asn Lys Thr Ile


    300                 305                 310


 


GTC TTT AAT CAA TCC TCA GGA GGG GAC CCA GAA ATT GCA
972


Val Phe Asn Gln Ser Ser Gly Gly Asp Pro Glu Ile Ala


            315                 320


 


ACG CTC AGT TTT AAT TGT GGA GGG GAA TTT TTC TAC TGT
1011


Thr Leu Ser Phe Asn Cys Gly Gly Glu Phe Phe Tyr Cys


325                 330                 335


 


AAT TCA ACA CAA CTG TTT AAT AGT ACT TGG AAT AGT ACT
1050


Asn Ser Thr Gln Leu Phe Asn Ser Thr Trp Asn Ser Thr


        340                 345                 350


 


GGG TCA AAT AAC ACT AAA GGA AAT GAC ACA ATC ACA CTC
1089


Gly Ser Asn Asn Thr Lys Gly Asn Asp Thr Ile Thr Leu


                355                 360


 


CCA TGC AGA ATA AGA CAA ATT ATA AAC ATG TGG CAG AAA
1128


Pro Cys Arg Ile Arg Gln Ile Ile Asn Met Trp Gln Lys


    365                 370                 375


 


ATA GGA AAA GCA ATG TAT GCC CCT CCC ATC AAA GGG CAA
1167


Ile Gly Lys Ala Met Tyr Ala Pro Pro Ile Lys Gly Gln


            380                 385


 


ATT AGA TGT TCA TCA AAT ATT ACA GGG CTA ATA TTA ACA
1206


Ile Arg Cys Ser Ser Asn Ile Thr Gly Leu Ile Leu Thr


390                 395                 400


 


AGA GAT GGT GGT AAC AAC AAC ATG AGC AAG ACC ACC GAG
1245


Arg Asp Gly Gly Asn Asn Asn Met Ser Lys Thr Thr Glu


        405                 410                 415


 


ACC TTC AGA CCT GGA GGA GGA GAT ATG AOG GAC AAT TGG
1284


Thr Phe Arg Pro Gly Gly Gly Asp Met Arg Asp Asn Trp


                420                 425


 


AGA AGT GAA TTA TAT AAA TAT AAA GTA GTA AAA ATT GAA
1323


Arg Ser Glu Leu Tyr Lys Tyr Lys Val Val Lys Ile Glu


    430                 435                 440


 


CCA TTA GGA GTA GCA CCC ACC AGG GCA AAG AGA AGA GTG
1362


Pro Leu Gly Val Ala Pro Thr Arg Ala Lys Arg Arg Val


            445                 450


 


GTG CAG AGA GAA AAA AGA GCA GTG GGA ATA GGA GCT GTG
1401


Val Gln Arg Glu Lys Arg Ala Val Gly Ile Gly Ala Val


455                 460                 465


 


TTC CTT GGG TTC TTG GGA GCA TAA AGC TTC TAG A 1435


Phe Leu Gly Phe Leu Gly Ala Xaa Ser Phe Xaa


        470                 475         478


 








CLONE C17.3










    CTC GAG GTA CCT GTG TGG AAA GAA GCA ACC ACC ACT
36



    Leu Glu Val Pro Val Trp Lys Glu Ala Thr Thr Thr


      1               5                  10


 


CTA TTT TGT GCA TCA GAT GCT AAA GCA TAT GAT TCA GAG
75


Leu Phe Cys Ala Ser Asp Ala Lys Ala Tyr Asp Ser Glu


          15                 20                  25


 


GCA CAT AAT GTT TGG GCC ACA CAT GCC TGT GTA CCC ACA
114


Ala His Asn Val Trp Ala Thr His Ala Cys Val Pro Thr


                 30                  35


 


GAC CCC AAC CCA CAA GAA GTA GAA TTG GAA AAT GTG ACA
153


Asp Pro Asn Pro Gln Glu Val Glu Leu Glu Asn Val Thr


     40                  45                  50


 


GAA AAT TTT AAC ATG TGG AAA AAT AAC ATG GTA GAA CAG
192


Glu Asn Phe Asn Met Trp Lys Asn Asn Met Val Glu Gln


             55                  60


 


ATG CAT GGG GAT ATA ATT AGT TTA TGG GAT CAA AGC CTA
231


Met His Gly Asp Ile Ile Ser Leu Trp Asp Gln Ser Leu


 65                  70                  75


 


AAG CCA TGT GTA AAA TTA ACC CCA CTC TGT GTT ACG TTA
270


Lys Pro Cys Val Lys Leu Thr Pro Leu Cys Val Thr Leu


         80                  85                  90


 


AAT TGC ACT GAC CCA AAT GTT ACT AAT AGC GAG AGA ACG
309


Asn Cys Thr Asp Pro Asn Val Thr Asn Ser Glu Arg Thr


                 95                 100


 


ATA GAG GGG GGA GAA ATA AAA AAT TGC TCT TTC AAT ATC
348


Ile Glu Gly Gly Glu Ile Lys Asn Cys Ser Phe Asn Ile


    105                 110                 115


 


ACC ACA AAC ATA AGA GAT AGG TTT CAG AAA GAA TAT GCA
387


Thr Thr Asn Ile Arg Asp Arg Phe Gln Lys Glu Tyr Ala


            120                 125


 


CTT TTT TAT AAA CTT GAT GTA ATA CCA TTA GGT AAT GAT
426


Leu Phe Tyr Lys Leu Asp Val Ile Pro Leu Gly Asn Asp


130                 135                 140


 


AAT ACT AGC TAT AGG TTG ATA AGT TGT AAC ACC TCA GTC
465


Asn Thr Ser Tyr Arg Leu Ile Ser Cys Asn Thr Ser Val


        145                 150                 155


 


ATT ACA CAG GCC TGT CCA AAG GTA TCC TTT GAG CCA ATT
504


Ile Thr Gln Ala Cys Pro Lys Val Ser Phe Glu Pro Ile


                160                 165


 


CCC ATA CAT TAT TGT GCC CCG GCT GGT TTT GCG ATT CTA
543


Pro Ile His Tyr Cys Ala Pro Ala Gly Phe Ala Ile Leu


    170                 175                 180


 


AAG TGT AAA GAT AAG AAG TTC AAT GGA ACA GGA CCA TGT
582


Lys Cys Lys Asp Lys Lys Phe Asn Gly Thr Gly Pro Cys


            185                 190


 


ACA AAT GTC AGC ACA GTA CAA TGT ACA CAT GGA ATT AAG
621


Thr Asn Val Ser Thr Val Gln Cys Thr His Gly Ile Lys


195                 200                 205


 


CCA GTA GTA TCA ACT CAA CTG TTG TTA AAT GGC AGT CTA
660


Pro Val Val Ser Thr Gln Leu Leu Leu Asn Gly Ser Leu


        210                 215                 220


 


GCA GAA GAA GAC ATA GTA ATT AGA TCC GCC AAT CTC ACA
699


Ala Glu Glu Asp Ile Val Ile Arg Ser Ala Asn Leu Thr


                225                 230


 


GAC AAT GCT AAA AAC ATA ATA GTA CAG CTG AAT GAA TCT
738


Asp Asn Ala Lys Asn Ile Ile Val Gln Leu Asn Glu Ser


    235                 240                 245


 


GTA ACA ATG AAT TGT ACA AGA CCC AAC AAC AAT ACA ATG
777


Val Thr Met Asn Cys Thr Arg Pro Asn Asn Asn Thr Met


            250                 255


 


AAA AGT ATA CAT ATA GGA CCA GGC AGA GCA TTT TAT GCA
816


Lys Ser Ile His Ile Gly Pro Gly Arg Ala Phe Tyr Ala


260                 265                 270


 


ACA GGA AAC ATA ATA GGA GAT ATA AGA CAA GCA CAT TGT
855


Thr Gly Asn Ile Ile Gly Asp Ile Arg Gln Ala His Cys


        275                 280                 285


 


AAC ATT AGT GGA ACA AAA TGG AAT GAC ACT TTG AAA AAG
894


Asn Ile Ser Gly Thr Lys Trp Aen Asp Thr Leu Lys Lys


                290                 295


 


ATA GCT ATA AAA TTA AGA GAA CAA TTT AAT AAG ACA ATA
933


Ile Ala Ile Lys Leu Arg Glu Gln Phe Asn Lys Thr Ile


    300                 305                 310


 


GTC TTT AAT CAA TCC TCA GGA GGG GAC CCA GAA ATT GCA
972


Val Phe Asn Gln Ser Ser Gly Gly Asp Pro Glu Ile Ala


            315                 320


 


ACG CTC AGT TTT AAT TGT GGA GGG GAA TTT TTC TAC TGT
1011


Thr Leu Ser Phe Asn Cys Gly Gly Glu Phe Phe Tyr Cys


325                 330                 335


 


AAT TCA ACA CAA CTG TTT AAT AGT ACT TGG AAT AGT ACT
1050


Asn Ser Thr Gln Leu Phe Asn Ser Thr Trp Asn Ser Thr


        340                 345                 350


 


GGG TCA AAT AAC ACT AAA GGA AAT GAC ACA ATC ACA CTC
1089


Gly Ser Asn Asn Thr Lys Gly Asn Asp Thr Ile Thr Leu


                355                 360


 


CCA TGC AGA ATA AGA CAA ATT ATA AAC ATG TGG CAG AAA
1128


Pro Cys Arg Ile Arg Gln Ile Ile Asn Met Trp Gln Lys


    365                 370                 375


 


ATA GGA AAA GCA ATG TAT GCC CCT CCC ATC AAA GGG CAA
1167


Ile Gly Lys Ala Met Tyr Ala Pro Pro Ile Lys Gly Gln


            380                 385


 


ATT AGA TGT TCA TCA AAT ATT ACA GGG CTA ATA TTA ACA
1206


Ile Arg Cys Ser Ser Asn Ile Thr Gly Leu Ile Leu Thr


390                 395                 400


 


AGA GAT GGT GGT AAC AAC AAC ATG AGC AAG ACC ACC GAG
1245


Arg Asp Gly Gly Asn Asn Asn Met Ser Lys Thr Thr Glu


        405                 410                 415


 


ACC TTC AGA CCT GGA GGA GGA GAT ATG AGG GAC AAT TGG
1284


Thr Phe Arg Pro Gly Gly Gly Asp Met Arg Asp Asn Trp


                420                 425


 


AGA AGT GAA TTA TAT AAA TAT AAA GTA GTA AAA ATT GAA
1323


Arg Ser Glu Leu Tyr Lys Tyr Lys Val Val Lys Ile Glu


    430                 435                 440


 


CCA TTA GGA GTA GCA CCC ACC AGG GCA AAG AGA AGA GTG
1362


Pro Leu Gly Val Ala Pro Thr Arg Ala Lys Arg Arg Val


            445                 450


 


GTG CAG AGA GAA AAA AGA GCA GTG GGA ATA GGA GCT GTG
1401


Val Gln Arg Glu Lys Arg Ala Val Gly Ile Gly Ala Val


455                 460                 465


 


TTC CTT GGG TTC TTG GGA GCA TAA AGC TTC TAG A 1435


Phe Leu Gly Phe Leu Gly Ala Xaa Ser Phe Xaa


        470                 475         478









In addition to the listing in Table 1, FIG. 3 shows the alignment of the amino acid sequences of the clones of each of the seven isolates. Corresponding residues from various clones are in boxes. In the figure, the amino acid sequences are aligned against MN-rgp120 (SEQ. ID. NO: 41.
In one embodiment, a gp120 polypeptide of this invention has the same amino acid sequence as the sequence of one of the breakthrough isolates. In another embodiment, the amino acid sequence is truncated, as described in detail hereinafter. In another embodiment, a gp120 polypeptide sequence of this invention contains a substitution, insertion, or deletion (alteration) of one or more amino acids in the sequence of a breakthrough isolate. Usually, with the exception of amino acids that are not present in a truncated amino acid sequence and eliminate an epitope, a gp120 polypeptide of this invention will include alterations in the amino acid sequence of a breakthrough isolate that do not alter the polypeptide's ability to induce the same neutralizing antibodies as the amino acid sequence of the isolate.
In general, substitutions in the amino acid sequence of a gp120 polypeptide of this invention are conservative substitutions, particularly for amino acid residues in the V2, V3, and C4 domains of gp120, which domains contain neutralizing epitopes. However, non-conservative substitutions, particularly in domains that do not contain neutralizing epitopes are contemplated.
Conservative substitutions replace an amino acid with an amino acid of similar size and character. For example, a hydrophobic residue or hydrophilic residue is replaced with another hydrophobic residue or hydrophilic residue, respectively. Amino acids can be divided into the following groups: positively charged residues (K, R and H); negatively charged residues (D and E); amides (N and Q); aromatics (F, Y, and W); hydrophobics (P, G, A, V, L, I, and M); and uncharged residues (S and T). Usually, residues within a group are replaced with another member of the group.
In one embodiment, critical amino acid residues in the V2, V3, and C4 domains of gp120 are identical to the corresponding residues in a breakthrough-isolate sequence. Critical amino acid residues in the V2, V3, and C4 domains of gp120 are described in the experimental section. In another embodiment, all amino acid residues in the V2, V3, and C4 domains of gp120 are identical to corresponding residues in a breakthrough isolate sequence.
Oligonucleotide Encoding gp120 from Breakthrough Isolates
The present invention also provides novel oligonucleotides encoding gp120 from the breakthrough isolates which can be used to express gp120. An oligonucleotide of this invention encodes a polypeptide of this invention. The oligonucleotide can be DNA or RNA, usually DNA. Although numerous nucleotide sequences can encode the same amino acid sequence due to the degeneracy of the genetic code, conveniently, the oligonucleotides of this invention include a nucleotide sequence of a breakthrough isolate as illustrated in Table 1 (Sequence ID NOs: 2, 5, 8, 10, 12, 16, 19, 23, 25, 28, 31, 33, 36). Usually, an oligonucleotide of this invention is less than about 5 kilobases (kb), preferably less than about 3 kb.
To express the encoded amino acid sequence, the oligonucleotide can be inserted into a transcription unit. The transcription unit can be inserted into a plasmid for production of cell lines, inserted into a virus (e.g.; vaccinia) or can be used directly as a DNA vaccine. Suitable transcription units for production of vaccine proteins are well known. A preferred expression vector, designated psvI6B5, is illustrated in Sequence ID NO: 45. The vector includes an HSV-1 gDl signal sequence joined to a linker sequence. The gp120 nucleotide sequence to be expressed starts with the Kpn I site of the gene. Since all gp120 or gp160 sequences contain this site, any gp120 nucleotide sequence can be analogously inserted into the vector and expressed. The vector ends with a poly A tail from SV40.
In addition to being useful to express a polypeptide sequence of this invention, the oligonucleotides of this invention can also be used in diagnostics to detect HIV isolates. For example, the oligonucleotide or a portion thereof encoding a neutralizing epitope can be used in branched chain DNA diagnostics or as a probe in in situ hybridization studies.
Vaccine Preparation
A gp120 polypeptide of this invention from a selected breakthrough isolate(s) in a suitable carrier is used to make a subunit vaccine. The polypeptide can be used alone, but is generally administered in a multivalent subunit vaccine that includes gp120 MN. In addition to one or more gp120 polypeptides of this invention, the vaccine generally includes the MN polypeptide (hereinafter, MN-rgp120). The vaccine usually includes about 3 to about 5 different gp120 polypeptides, but 30 or more different gp120 polypeptides can be used.
Preparation of gp120 polypeptides for use in a vaccine is well known and is described hereinafter. With the exception of the use of the selected HIV isolate, the gp120 subunit vaccine prepared in the method does not differ from gp120 subunit vaccines of the prior art.
As with prior art gp120 subunit vaccines, gp120 at the desired degree of purity and at a sufficient concentration to induce antibody formation is mixed with a physiologically acceptable carrier. A physiologically acceptable carrier is nontoxic to a recipient at the dosage and concentration employed in the vaccine. Generally, the vaccine is formulated for injection, usually intramuscular or subcutaneous injection. Suitable carriers for injection include sterile water, but preferably are physiologic salt solutions, such as normal saline or buffered salt solutions such as phosphate-buffered saline or ringer's lactate. The vaccine generally contains an adjuvant. Useful adjuvants include QS21 (Quillaja saponaria, commercially available from Cambridge Biotech, Worcester, Mass.), which stimulates cytotoxic T-cells, and alum (aluminum hydroxide adjuvant). Formulations with different adjuvants which enhance cellular or local immunity can also be used. In particular, immunopotentiators such as cytokines can be included in the vaccine. Examples of suitable immunopotentiating cytokines include interleukins, such as interleukin-2 (IL-2) and interleukin-12 (IL-12), and tumor necrosis factor-alpha (TNF-α).
Additional excipients that can be present in the vaccine include low molecular weight polypeptides (less than about 10 residues), proteins, amino acids, carbohydrates including glucose or dextrans, chelating agents such as EDTA, and other excipients that stabilize the protein or inhibit growth of microorganisms.
The vaccine can also contain other HIV proteins. In particular, gp41 or the extracellular portion of gp41 or HIV-1 core proteins such as P24, P17, and P55 can be present in the vaccine. Although the amino acid sequence of gp41 is more conserved than that of gp120, gp41 contains neutralizing epitopes. Preferably, any gp41 present in the vaccine is from an HIV isolate present in the vaccine. gp160 from an isolate used in the vaccine can replace gp120 in the vaccine or be used together with gp120 from the isolate. Alternatively, gp160 from a different isolate than those in the vaccine can additionally be present in the vaccine.
Vaccines according to the invention can also contain one or more soluble gp120 polypeptide sequences, or fragments thereof, in combination with an engineered virus specifically designed to express proteins that induce a cytotoxic T-cell response. Suitable engineered viruses are derived from, for example, Canary Pox virus, vaccinia viruses, attenuated human herpes viruses (such as, e.g., herpes simplex viruses), and Varicella Zoster. Exemplary engineered viruses are modified to express any HIV protein capable of inducing a cytotoxic T-cell response, such as those described above. Typically, immunization with the gp120/engineered virus vaccine is followed by administration of one or more doses of the gp120 polypeptide sequence(s) to boost the immune response. If desired, viruses can be engineered to express one or more gp120 polypeptide sequences of the invention, or fragments thereof, and used in vaccines with or without soluble gp120 polypeptide sequences.
Vaccine formulations generally include a total of about 300 to 600 .mu.g of gp120, conveniently in about 1.0 ml of carrier. Preferred formulations include use of twice the weight of a gp120 polypeptide in twice as 600 .mu.g alum. However, formulations having smaller amounts (e.g.; 50 .mu.g per dose) are also used, generally with alum or other adjuvants. The amount of gp120 for any isolate present in the vaccine will vary depending on the immunogenicity of the gp120. For example, gp120 from some strains of HIV may be less immunogenic than gp120 from the MN strain (Sequence ID NO: 41). If two strains having different immunogenicity are used in combination, empirical titration of the amount of each virus would be performed to determine the percent of the gp120 of each strain in the vaccine. For isolates having similar immunogenicity, approximately equal amounts of each isolate's gp120 would be present in the vaccine. For example, in a preferred embodiment, the vaccine includes gp120 from the MN and a strain of this invention at concentrations of about 300 .mu.g per strain in about 1.0 ml of carrier. When the vaccine includes gp120 from about 30 isolates, about 10 to about 50 .mu.g can be used. Methods of determining the relative amount of an immunogenic protein in multivalent vaccines are well known and have been used, for example, to determine relative proportions of various isolates in multivalent polio vaccines.
The vaccines of this invention are administered in the same manner as prior art HIV gp120 subunit vaccines. In particular, the vaccines are generally administered at 0, 1, and at 6, 8 or 12 months, depending on the protocol. A preferred protocol includes administration at 0, 1, 6, and 12 months. Following the immunization procedure, annual or bi-annual boosts can be administered. However, during the immunization process and thereafter, neutralizing antibody levels can be assayed and the protocol adjusted accordingly.
The vaccine is administered to uninfected individuals. In addition, the vaccine can be administered to seropositive individuals to augment immune response to the virus, as with prior art HIV vaccines. It is also contemplated that DNA encoding the strains of gp120 for the vaccine can be administered in a suitable vehicle for expression in the host. In this way, gp120 can be produced in the infected host, eliminating the need for repeated immunizations. Preparation of gp120 expression vehicles is described hereinafter.
Although the gp120 isolates described herein can be used as a vaccine as described above, the amino acid sequences can also be used alone or in combinations in the same type of formulation for use as an immunogen, to induce antibodies that recognize the isolate(s) present in the immunogen. Immunogens are formulated in the same manner as vaccines and can include the same excipients, etc. Antibodies induced by the immunogens can be used in a diagnostic to detect the HIV strain in the immunogen or to affinity purify the strain.
gp120 Polypeptide Sequences and Chemokine Receptors
While CD4 is the primary cellular receptor for HIV-1, it is not sufficient for entry of HIV-1 into cells. Co-receptors required in conjunction with CD4 have been identified. These co-receptors are members of the chemokine receptor family of seven-transmembrane G-protein coupled receptors. The chemokine superfamily is subdivided into two groups based on the amino terminal cysteine spacing. The CXC chemokines are primarily involved in neutrophil-mediated inflammation, and the CC chemokines tend to be involved in chronic inflammation. At least five CC chemokine receptors, designated CC-CKR1–5 (also known in the art as CCR1–5), and at least four CXC chemokine receptors, designated CXC-CKR1–4 (also known as CXCR-1–4), have been identified.
CXC-CKR-4 (CXCR-4), which has also been called the alpha-chemokine receptor fusin, serves as an entry cofactor for T-cell-tropic HIV-1 strains. CC-CKR-5 (CC-RS), which has been called beta-chemokine receptor, together with its related family members, such as CC-CKR-2b and CC-CKR3, serve as entry cofactors for macrophage-tropic HIV-1 strains. T-cell-tropic strains can infect primary T-cells and T-cell lines, but not macrophages, whereas macrophage-tropic strains can infect macrophages and primary T-cells, but not T-cell lines. T-cell- and macrophage-tropic strains are discussed more fully in Deng et. al., Nature 381:661–666 (1996), which is hereby incorporated by reference in its entirety. Examples of T-cell-tropic strains include laboratory isolates, such as IIIB and MN. Macrophage-tropic strains include primary isolates, including but not limited to CM244, GNE6, GNE8, and breakthrough viruses from vaccinees immunized with gp120-based vaccines. Dual-tropic strains can, use both types of co-receptors, entering cells via CXC-CKR-4 or via one or more CC-CKR family members, preferably CC-CKR-5, CC-CKR-2b, or CC-CKR-3. While the present invention is not intended to be bound or limited by any one theory, the entry of T-cell tropic and macrophage-tropic HIV-1 strains is believed to provide a unifying explanation of the differences in cell tropism between viral strains, the resistance to HIV-1 infection by many CD4-transfected nonprimate cells, and the HIV-1-infection resistance of a portion of the human population.
Accordingly, in one embodiment is a vaccine containing (1) a first gp120 polypeptide sequence, or fragment thereof, from a macrophage-tropic HIV-1 strain and/or a second gp120 polypeptide sequence, or fragment thereof, from a T-cell tropic strain, in combination with (2) a breakthrough isolate HIV gp120 polypeptide sequence, or fragment thereof, from a vaccinee vaccinated with the first and/or second HIV gp120 polypeptide sequence. Preferably, the vaccine includes at least two gp120 polypeptide sequences that bind to different chemokine receptors. In one embodiment, the vaccine includes first and second gp120 polypeptide sequences that bind to different chemokine receptors. In addition, the breakthrough isolate gp120 polypeptide sequence can bind to a different chemokine receptor than the chemokine receptor(s) bound by either or both of the first and second gp120 polypeptide sequence(s).
A preferred T-cell tropic strain is a laboratory isolate, most preferably MN. Preferred macrophage-tropic viruses for use in the invention are GNE6 and GNE8, which are representative of the breakthrough viruses disclosed herein and differ from MN in that their gp120s induce the formation of antibodies that recognize the gp120 sequences (e.g., the V3 domain) involved in binding to CC chemokine receptors, such as CXC-CKR-5.
In one embodiment, HIV infection is prevented by administering one or more chemokine receptor-binding gp120 polypeptide sequences, or fragment(s) thereof containing appropriate chemokine receptor-binding domains, in a vaccine, such as those described above. Preferably, the vaccine also includes one or more CD4-binding gp120 polypeptide sequences or appropriate fragments thereof. Such vaccines induce anti-HIV antibodies that inhibit viral gp120-chemokine receptor or -CD4 binding. In addition, such gp120 polypeptides can directly inhibit HIV infection by binding to one or more co-receptors for HIV infection, such as CD4 or a chemokine receptor, thus providing a prophylactic or therapeutic effect in treating HIV infection. Preferably, gp120 polypeptide sequences useful in this regard contain the T-cell binding (TCB) domain.
Various uses of chemokine receptor-binding gp120 polypeptides are discussed below with regard to the CC chemokine receptor family. However, those skilled in the art recognize that this discussion applies equally to CXC chemokine receptors that act as cofactors in HIV infection.
The gp120 polypeptides can be used as a composition containing one or more gp120 polypeptides, as described for use as a vaccine or immunogen. The composition can be administered, prophylactically or therapeutically, to a patient at risk of infection or in need of such treatment using the dosages and routes and means of administration described herein. However, chronic administration may be preferred and dosages can be adjusted accordingly. It is noted that in vivo administration can also induce antibodies that bind viral gp120, further inhibiting virus binding to CC-CKR.
The gp120 polypeptides can also be used in screening assays to identify antagonists of CC-CKR. For example, candidate antagonists can be screened for inhibition of binding of gp120 to a CC-CKR CC-CKR receptor that is isolated and attached to a surface (e.g., plastic dish) or recombinantly or naturally expressed on the surface of a cell. Antagonists can either bind gp120 or bind receptor. Preferred candidate antagonists include gp120 compounds, small gp120 peptides (5 to 20 amino acids in length, preferably 7 to 10 amino acids in length) or peptidomimetics of gp120 that bind receptor, monoclonal antibodies that bind gp120, and small organic molecules that bind either gp120 or receptor.
The antibodies induced by the gp120 polypeptides can also be used to induce anti-idiotype antibodies that bind CC chemokines. These anti-idiotype antibodies can be screened for binding to an anti-gp120 polypeptide antibody and inhibiting gp120 from binding CC-CKR receptor. Such anti-idiotype antibodies mimic gp120 by binding to CC-CKR receptor. Such antibodies, preferably human antibodies, can be obtained in a number of ways, such as human antibodies from combinatorial libraries (e.g., Burton et al. Adv. Immunolo. (1994) 57:191–280). It is now possible to produce transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, homozygous deletion of the antibody heavy-chain joining region (JH) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array in such germ-line mutant mice results in the production of human antibodies upon antigen challenge as described in Jakobovitis et al., Proc. Natl. Acad. Sci. USA 90: 2551 (1993); Jakobovits et al., Nature 362:255–258 (1993); Bruggermann et al., Year in Immuno. 7: 33 (1993).
Alternatively, phage display technology as described by McCafferty et al., Nature 348:552–553 (1990) can be used to produce human antibodies and antibody fragments in vitro from immunoglobulin variable (V) domain gene repertoires from unimmunized donors. According to this technique, antibody V domain genes are closed in-frame either into either a major or minor coat protein gene of a filamentous bacteriophage, such as M13 or fd, and displayed as functional antibody fragments on the surface of the phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody also result in selection of the gene encoding the antibody exhibiting those properties. Phage display can be performed in a variety of formats as reviewed by, for example, Johnson, et al., Current Opinion in Structural Biology 3:564–571 (1993).
Several sources of V-gene segments can be used for phage display. Clackson et al., Nature, 352: 624–628 (1991) isolated a diverse array of anti-oxazolone antibodies from a small random combinatorial library of V genes derived from the spleens of immunized mice. A repertoire of V genes from unimmunized human donors (or embryonic cells) can be constructed. It has been demonstrated that antibodies to a diverse array of antigens (including self-antigens) can be isolated essentially following the techniques described by Marks et al., J. Mol. Biol., 222: 581–597 (1991), or Griffith et al., EMBO J., 12: 725–734 (1993).
In a natural immune response, antibody genes accumulate mutations at a high rate (somatic hypermutation). Some of the changes introduced confer higher affinity, and B cells displaying high-affinity surface immunoglobulin are preferentially replicated and differentiated during subsequent antigen challenge. This natural process can be mimicked by employing the technique known as “chain shuffling” (Marks et al., Bio/Technol. 10:779–783 [1992]). In this method, the affinity of “primary” human antibodies obtained by phage display can be improved by sequentially replacing the heavy and light chain V region genes with repertoires of naturally occurring variants (repertoires) of V domain genes obtained from unimmunized donors. This technique allows the production of antibodies and antibody fragments with affinities in the nM range. A strategy for making very large phage antibody repertoires has been described by Waterhouse et al., Nucl. Acids Res., 21: 2265–2266 (1993).
Accordingly, antibodies that bind CC-CKR can be obtained by screening antibodies or fragments thereof expressed on the surface of bacteriophage in combinatorial libraries or in other systems as described above with a gp120 monoclonal antibody that inhibits gp120 binding to receptor.
In addition to screening antibodies with a gp-120 antibody, random or combinatorial peptide libraries can be screened with either a gp120 antibody or the gp120 compounds of the invention. Approaches are available for identifying peptide ligands from libraries that comprise large collections of peptides, ranging from 1 million to 1 billion difference sequences, which can be screened using monoclonal antibodies or target molecules. The power of this technology stems from the chemical diversity of the amino acids coupled with the large number of sequences in a library. See for example, Scott et al., Cur. Open Biotechnol. 5(1):40–8 (1994); Kenan et al. Trends Biochem. Sci. (1994) 19(2):57–64. Accordingly, the monoclonal antibodies, preferably human monoclonals or fragments thereof, generated as discussed herein, find use in treatment by inhibiting or treating HIV infection or disease progression, as well as in screening assays to identify additional pharmaceuticals.
Production of gp120
gp120 for a vaccine can be produced by any suitable means, as with prior art HIV gp120 subunit vaccines. Recombinantly-produced or chemically synthesized gp120 is preferable to gp120 isolated directly from HIV for safety reasons. Methods for recombinant production of gp120 are described below.
Oligonucleotides encoding gp120 from breakthrough isolates and capable of expressing gp120 can be prepared by conventional means. For example, the nucleotide sequence can be synthesized. Alternatively, another HIV nucleotide sequence encoding gp120 can be used as a backbone and altered at any differing residues as by site-directed mutagenesis. Site-directed mutagenesis is described in Kunkel et al, Proc. Natl. Acad. Sci. (USA) 82:488–492 (1985) and Zoller et al, Nuc. Acids Res. 10:6487–6500 (1982) and is well known.
In a preferred embodiment, the nucleotide sequence is present in an expression construct containing DNA encoding gp120 under the transcriptional and translational control of a promoter for expression of the encoded protein. The promoter can be a eukaryotic promoter for expression in a mammalian cell. In cases where one wishes to expand the promoter or produce gp120 in a prokaryotic host, the promoter can be a prokaryotic promoter. Usually a strong promoter is employed to provide high-level transcription and expression.
The expression construct can be part of a vector capable of stable extrachromosomal maintenance in an appropriate cellular host or may be integrated into host genomes. Normally, markers are provided with the expression construct which allow for selection of a host containing the construct. The marker can be on the same or a different DNA molecule, desirably, the same DNA molecule.
The expression construct can be joined to a replication system recognized by the intended host cell. Various replication systems include viral replication systems such as those from retroviruses, simian virus, bovine papilloma virus, or the like. In addition, the construct may be joined to an amplifiable gene, e.g. the DHFR gene, so that multiple copies of the gp120 DNA can be made. Introduction of the construct into the host will vary depending on the construct and can be achieved by any convenient means. A wide variety of prokaryotic and eukaryotic hosts can be employed for expression of the proteins.
Preferably, the gp120 is expressed in mammalian cells that provide the same glycosylation and disulfide bonds as in native gp120. Expression of gp120 and fragments of gp120 in mammalian cells as fusion proteins incorporating N-terminal sequences of Herpes Simplex Virus Type 1 (HSV-1) glycoprotein D (gD-1) is described in Lasky, L. A. et al., 1986 (Neutralization of the AIDS retrovirus by antibodies to a recombinant envelope glycoprotein) Science 233: 209–212 and Haffar, O. K. et al., 1991 (The cytoplasmic tail of HIV-1 gp160 contains regions that associate with cellular membranes.) Virol. 180:439–441, respectively. A preferred method for expressing gp120 is described in the examples. In the examples, a heterologous signal sequence was used for convenient expression of the protein. However, the protein can also be expressed using the native signal sequence.
An isolated, purified gp120 polypeptide having one of the amino acid sequences illustrated in Table 1 can be produced by conventional methods. For example, the proteins can be chemically synthesized. In a preferred embodiment, the proteins are expressed in mammalian cells using an expression construct of this invention. The expressed proteins can be purified by conventional means. A preferred purification procedure is described in the examples.
gp120 Fragments
The present invention also provides gp120 fragments that are suitable for use in inducing antibodies for use in a vaccine formulation. A truncated gp120 sequence, as used herein, is a fragment of gp120 that is free from a portion of the intact gp120 sequence beginning at either the amino or carboxy terminus of gp120. A truncated gp120 sequence of this invention is free from the Cs domain. The CS domain of gp120 is a major immunogenic site of the molecule. However, antibodies to the region do not neutralize virus. Therefore, elimination of this portion of gp120 from immunogens used to induce antibodies for serotyping is advantageous.
In another embodiment, the truncated gp120 sequence is additionally free from the carboxy terminal region through about amino acid residue 453 of the gp120 V5 domain. The portion of the V5 domain remaining in the sequence provides a convenient restriction site for preparation of expression constructs. However, a truncated gp120 sequence that is free from the entire gp120 V5 domain is also suitable for use in inducing antibodies.
In addition, portions of the amino terminus of gp120 can also be eliminated from the truncated gp120 sequence. In particular, the truncated gp120 sequence can be free from the gp120 signal sequence. The truncated gp120 sequence can be free from the carboxy terminus through amino acid residue 111 of the gp120 C1 domain, eliminating most of the C1 domain but preserving a convenient restriction site. However, the portion of the C1 domain through the V2 cysteine residue that forms a disulfide bond can additionally be removed, so that the truncated gp120 sequence is free from the carboxy terminus through amino acid residue 117 of the gp120 C1 domain. In a preferred embodiment, the truncated gp120 sequence is free from the amino terminus of gp120 through residue 111 of the C1 domain and residue 453 through the carboxy terminus of gp120.
The truncated gp120 sequences can be produced by recombinant engineering, as described previously Conveniently, DNA encoding the truncated gp120 sequence is joined to a heterologous DNA sequence encoding a signal sequence.
It is understood that the application of the teachings of the present invention to a specific problem or situation is within the capabilities of one having ordinary skill in the art in light of the teachings contained herein. Examples of the products of the present invention and representative processes for their isolation, use, and manufacture appear-below, but should not be construed to limit the invention. All literature citations herein are expressly incorporated by reference.
EXAMPLES
Materials and Methods
Specimen collection from human volunteers. Blood was collected from MN-rgp120-immunized individuals who were infected with HIV-1 while participating in Phase I (NIH Protocol AVEG 016) and Phase II (NIH Protocol AVEG 201) HIV-1 vaccine trials sponsored by the National Institutes of Health (NIH). The demographics of the subjects in the study, and the study design have been described in McElrath; Seminars in Cancer Biol. 6:1–11 (1995); McElrath et al.; Abstracts from Eighth Annual Meeting of the National Cooperative Vaccine Development Groups for AIDS. Bethseda, Md. 216 (1996). Specimens were obtained according to an informed consent protocol approved by the institutional review boards of the participating institutions. In the experimental section, the time of HIV-1 infection is specified with regard to data provided by the NIH AIDS Vaccine Evaluation Network where PCR (RNA) and/or serologic assays were used to detect HIV-1 infection.
Sample preparation for cloning HIV-1 envelope glycoproteins. Peripheral blood mononuclear cells (PBMCS) from HIV-1 infected vaccinees were prepared from heparinized venous blood by FICOLL-HYPAQUE gradient centrifugation. Cell number and viability were determined. After separation, PBMCs were washed twice in phosphate-buffered saline and suspended at a cell density of 6×106 cells/ml in PCR lysis buffer (50 mM KCl, 10 mM Tris (pH 8.4), 2.5 mM MgCl2, 0.1 mg/ml gelatin (Sigma), 0.45% NONIDET P40 detergent, 0.45% TWEEN 20 detergent (both detergents are commercially available from United States Biochemical Corp.) and 0.06 mg/ml Proteinase K (Gibco BRL) to lyse the cells. The lysate was incubated at 50–60° C. for 1 hour, followed by inactivation of the Proteinase K at 95° C. for 10 minutes. Lysates were shipped frozen and stored at −70° C. until use.
Polymerase chain reaction (PCR) amplification. Samples were subjected to two rounds of PCR amplification using the nested primers described below. In the first round, 25 μl aliquots of PBMC lysates (containing about 1 μg genomic DNA) were mixed with an equal volume of a PCR reaction mix containing 400 μM each dNTP, 200 μg/ml BSA (Sigma Chemical Corporation, RIA grade) and about 100 pmoles of each primer in 50 mM KCl, 20 mM Tris (pH 8.4) and 3 mM MgCl2. After an initial 10 minute denaturation step at 95° C., 5 units of Taq polymerase (AMPLITAQ, Perkin Elmer Cetus) were added during an 55° C. soak step, and samples were overlayed with mineral oil.
The PCR profile was as follows: 2 cycles having 1 minute at 55° C., 2.5 minutes at 72° C. and 1 minute at 94° C., followed by 28 cycles with 30 seconds at 55° C., 2.5 minutes at 72° C. and 45 seconds at 94° C., and an extension step at 72° C. for 5 minutes.
Aliquots of 10 .mu.l from the first-round reactions were re-amplified with appropriate nested primers in a final reaction volume of 100 .mu.l, using either the reagents and profile described above or the reagents and profile described in the PCR Optimizer Kit (Invitrogen.) PCR reaction products were purified using QIAQUICK-spin columns (Qiagen Inc.) The primer pair used in the first round was either 120.os.F (5′-gggaattcggatccAGAGCAGAAGACAGTGGCAATGA with homologous sequence at position 6248–6270 of HIVPV22) (SEQ. ID. NO: 47) or JM11A (5′-ctcgag-CTCCTGAAGACAGTCAGACTCATCAAG at position 6048–6074) (SEQ. ID. NO: 48) in the forward direction [Kusumi et al.; J. Virol. 66:875 (1992)] combined with 120.os.R (5′-ggtctagaagctttaGCCCATAGTGCTCCTGCTGCT-CC at position 7836–7859) (SEQ. ID. NO: 49) in the reverse direction. The internal nested primers were 120.BX.F (5′-gggcggatcctcgaGGTACCTGTRTGGAAAG-AAGCA at position 6389–6410; R: A or G) (SEQ. ID. NO: 50) and 120.is.R (5′-ggtctagaagctttaTGCTCCYAAGAACCCAAGGAACA at position 7819–7841; Y: T or C) (SEQ. ID. NO: 51). Heterologous primer sequences are shown in lower case letters.
Subcloning of PCR products and the expression of recombinant envelope glycoproteins as fusion proteins. The HIV-1 envelope glycoprotein gp120 sequences were cloned and expressed as chimeric genes and fusion proteins, where the signal sequence and 27 amino acids from the mature N terminus of herpes simplex virus type 1 (HSV-1) were fused to the N-terminal sequences of the gp120 genes, corresponding to amino acid 13 of the mature gp120 sequence. PCR products containing gp120 sequences from the breakthrough specimens were cloned into pRK5 expression plasmid as chimeric genes using combinations of restrictions sites engineered into the heterologous PCR primer tails and the Xho I site engineered into the N-terminal sequence of HSV-1 gD.
The resulting double-stranded DNA was sequenced with Sequenase and the dGTP Reagent Kit (United States Biochemical Corp.). Sequences from glycoprotein D were provided to enhance expression and to provide a flag epitope to facilitate protein analysis, as described in Berman et al.; J. Virol. 7:4464–9 (1992); Nakamura et al.; AIDS and Human Retroviruses 8:1875–85 (1992); and Nakamura et al.; J. Virol. 67:6179–91 (1993).
Briefly, isolated DNA fragments generated by the PCR reaction were ligated into a plasmid (pRK.gD-5, pRKgDstop) designed to fuse the gp120 fragments, in frame, to the 5′ sequences of the glycoprotein D (gD) gene of Type 1 Herpes Simplex Virus (gD-1) and the 3′ end to translational stop codons. The fragment of the gD-1 gene encoded the signal sequence and 25 amino acids of the mature form of HSV-1 protein. To allow for expression in mammalian cells, chimeric genes fragments were cloned into the pRK5 expression plasmid (Eaton et al., Biochemistry 291:8343–8347 (1986)) that contained a polylinker with cloning sites and translational stop codons located between a cytomegalovirus promotor and a simian virus 40 virus polyadenylation site.
The resulting plasmids were transfected into the 293s embryonic human kidney cell line (Graham et al., J. Gen. Virol. 36:59–77 (1977)) using a calcium phosphate technique (Graham et al., Virology 52:456–467 (1973)). Growth conditioned cell culture media was collected 48 hr after transfection, and the soluble proteins were detected by ELISA or by specific radioimmunoprecipitation where metabolically labeled proteins from cell culture supernatants were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (PAGE) and visualized by autoradiography as described in Berman et al., J. Virol. 63:3489–3498 (1989) and Laemmli Nature 227:680–685 (1970).
Serologic assays. Sera were assayed for antibodies to rgp120, antibodies to synthetic gp120 V3 domain peptides corresponding to sequences from the gp120 V3 domain, and antibodies able to inhibit the binding of MN-rgp120 to cell surface CD4 using serologic assays described in Berman et al.; J. Virol. 7:4464–9 (1992); Nakamura et al.; AIDS and Human Retroviruses 8:1875–85 (1992); and Nakamura et al.; J. Virol. 67:6179–91 (1993). Endpoint titers of antibody binding to gp120 and V3 peptides were determined using three fold-serial dilutions of sera. The endpoint dilution titer was defined as the last dilution that produced an optical density value that was two times higher than the mean of the optical densities of 1:50 diluted, pooled, normal human sera. Antibody titers were calculated by a computer program that interpolated values between antibody dilutions. The inter-assay coefficient of variation of positive control standard sera was 35%.
Binding of monoclonal antibodies to rgp120 from breakthrough viruses. An ELISA similar to that described by Moore et al.; AIDS 3:155–63 (1989) was used to measure the binding of various monoclonal antibodies (MAbs) to rgp120s from breakthrough viruses. Briefly, Nunc-Immuno plates (Maxisorp, certified) were coated (100 μl at 5 μg/ml in PBS at 4° C. overnight) with an affinity-purified sheep polyclonal antiserum to a peptide at the C terminus of gp120 (D7324, International Enzymes, Fallbrook, Calif.). After washing once with PBS-0.05% TWEEN-20 detergent, the plates were blocked with PBS-1.0% BSA for 30–60 minutes at room temperature. Cell culture supernatants from 293s cells, diluted to contain equivalent amounts of the gD-rgp120 fusion protein, were added and incubated for 2 hours at room temperature followed by three washes with PBS-0.05% TWEEN-20 detergent. Various MAbs were diluted in PBS-1.0% BSA and 100 μL of the diluted MAbs were added to each well and incubated for 1 hour at room temperature.
The plates were washed 3 times and incubated with 100 μl of a horseradish peroxidase-conjugated second antibody (goat anti-mouse or anti-human IgG, Cappel) for 1 hour at room temperature. After 3 washes the plates were developed and the OD492 (optical density at 492 nm) read in a plate reader. Growth conditioned cell culture supernatants were normalized by dilution based on binding by MAb 5B6 which is specific for HSV-1 glycoprotein D fusion protein.
Virus neutralization assays. The ability of vaccinee sera to inhibit infection of MT4 cells by HIV-1MN was measured in a cytopathicity assay where cell viability was quantitated using a calorimetric indicator dye, as described in Robertson et al.; J. Virol. Methods 20:195–202 (1988). Briefly, a virus stock of HIV-1MN (obtained from Dr. Michael Norcross, U.S. Food and Drug Administration) was prepared as the clarified supernatant from chronically infected H9/HIV-1MN cell culture. H9 cells chronically infected with HIV-MN were pelleted and resuspended in one-tenth the original volume of medium. Cell-associated virus was released by the mechanical shearing effects of rapid vortexing of the cells as described in Wrin et al.; J. Virol. 69:39–48 (1995).
An amount of virus sufficient to ensure complete cell lysis killing in 7 days was incubated with three-fold serial dilutions of test antisera, and then used to challenge MT4 T-lymphoid cells in 10% FCS/RPMI-1640 cell culture media. The cultures were incubated for 7 days at 37° C. in 5% CO2, and then cell viability was tested by the dye MTT, as described by Robertson et al.; J. Virol. Methods 20:195–202 (1988). Virus neutralization endpoints were quantitated by measurement of OD at 570–650 nm, and then the endpoint titers were calculated as the reciprocal of the antiserum dilution giving a signal that was two-fold above the control signal with unprotected (killed) cells. These titers were typically twice those calculated at 50% protection.
Results
Immunization history of infected subjects. Since 1992, 499 adults have been immunized with MN-rgp120 in Phase I trials in low or moderate risk individuals and in a Phase II clinical trial involving moderate to high risk individuals. The studies described herein entail the genetic and immunologic characterization of the first seven of nine individuals who became infected with HIV-1 through high risk behavior during the course of these trials. A listing of the trials and summary of the status of the vaccinees is presented in Table 2A. A listing of the analysis of the vaccinees is presented in Table 2B.







TABLE 2A







Description of Vaccinees Infected with HIV-1


After Immunization with MN-rgp120













‡Antigen dose/


Study No.
Case No.
*Risk Group
Adjuvant





016
C6
M/H
300/QS21


016
C8
M/H
600/QS21


016
C15
M/H
300/QS21


201
C7
M/H
600/Alum


201
C11
M/H
600/Alum


201
C10
M/IDU
600/Alum


201
C17
M/IDU
600/Alum





*M/H indicates male homosexual; M/IDU indicate male intravenous drug user.


‡numbers indicate dose in micrograms of MN-rgp120 injected per immunization; QS21 indicates antigen was formulated in QS21 adjuvant; Alum indicates MN-rgp120 formulated in aluminum hydroxide.













TABLE 2B







Description of Vaccinees Infected with HIV-1


After Immunization with MN-rgp120














Injection
Injections
Time of
 Interval:



Case
Schedule
before
HIV-1+
to HIV-1+



No.
(months)
HIV-1+
(months)
(months)

















C6
0, 1, 10.5
2
4.00
2.00



C8
0, 1
2
4.00
3.00



C15
0, 1, 2
3
6.25
4.00



C7
0, 1, 6, 12
3
9.25
3.00



C11
0, 1, 6, 12
4
19.50
6.75



C10
0, 1, 6, 19
3
19.50
13.50



C17
0, 1, 6, 18
4
24.75
6.25







 indicates interval between last immunization and detection of HIV-1 infection.






Three of the infections occurred in a Phase I trial (NIH Protocol AVEG 201) that compared the safety and immunogenicity of MN-rgp120 formulated in two different adjuvants (alum and QS21), and four of the infections occurred in a Phase II trial aimed at establishing the safety and immunogenicity of MN-rgp120 in various high risk groups (e.g., intravenous drug users, homosexual and bisexual males, and partners of HIV-1 infected individuals).
Of the seven infections studied (Table 3), two (C6 and C8) occurred after two injections, three (C7, C10 and C15) occurred after three injections, and two (C11 and C17) occurred after receiving the four scheduled injections. The interval between receiving the last immunization and becoming infected was 2 to 13.5 months.







TABLE 3







Peak Post Boost MN-rgp120 Antibody Titers


in Vaccinees that Became Infected with HIV-1














Injections
C6
C8
C15
C7
C11
C10
C17

















1
<50
2185
79
<50
1890
na
na


2
21539
10125
na
413
32696
7771
7056


3
#
#
4460
9707
34728
11627
18413


4
#
#
#
#
#
#
11340





# indicates specimen not analyzed because of HIV-1 infection.


na indicates the sample was not available for testing.


boldface indicates unusually low antibody titers.






Antibody response to gp120 in vaccinated individuals. The magnitude and specificity of the antibody response to MN-rgp120 was measured by ELISA in all infected individuals throughout the course of the immunization regime (FIG. 1). Five of the seven subjects exhibited normal antibody response kinetics that included a small but reproducible primary response (1:100–1:2,000) and a strong secondary (booster) response (titters ranging from 1:7,000–1:32,000), and antibody responses following third and fourth injections that were similar or marginally higher than those achieved after the second immunization (FIG. 1, Table 3).
The antibody response observed in C7 (FIG. 1C) was unusual in that no antibodies were detectable after the primary injection and a titer of only 1:350 was detected after the second injection. It thus appeared that C7 did not respond to the primary immunization, and that the antibody response obtained after the second injection represented a primary immune response. Consistent with this hypothesis, the third injection elicited a titer of only 1:9,707, typical of those normally seen after two immunizations.
An a typical antibody response was also seen in subject C15 (FIG. 1G) who was immunized according to an accelerated immunization schedule of 0, 1, and 2 months. As expected, the antibody titer seen in this subject (1:4,460) was at the low end of what is typically achieved after two immunizations and was far below normal values for three immunizations. The lack of an effective booster response after the third immunization of C15 was not surprising in view of previous studies where an accelerated 0, 1, and 2 month immunization schedule in baboons [Anderson et al.; J. Infect. Dis. 160:960–9 ((1989)] similarly prolonged the secondary response and failed to elicit an effective tertiary booster response.
Retrospective analysis of serum and plasma from subjects C6 (FIG. 1A) and C8 (FIG. 1B) indicated that they became infected with HIV-1 at some point between the second and third immunizations. Serologic evidence of HIV-1 infection was evident in the gp120 antibody assays where the titers failed to decline two weeks after the second injection and instead formed an uncharacteristic high titer plateau (FIGS. 1A and 1B). A similar plateau in MN-rgp120 titer after the third injection, suggested that subject C7 became infected around week 36, approximately 16 weeks after receiving the third injection (FIG. 1C). Subjects C10 (FIG. 1E), C11 (FIG. 1D), C15 (FIG. 1G), and C17 (FIG. 1F) developed unexpected increases in gp120 titers, typical of HIV-1 infection, after either the third or fourth immunizations. The data obtained demonstrate that immunologic priming for MN-rgp120 antibody responses is insufficient to provide universal protection from HIV-1 infection.
Antibody titers to the V3 domain. To further characterize the antibody response to gp120, antibody titers were measured to a synthetic V3 domain peptide of MN-rgp120 containing the principal neutralizing determinant (PND). Five of the seven subjects developed good V3 titers (1:400 to 1:4000) after the second immunization, however two subjects (C7 and C15) required three immunizations before developing significant tiers (FIGS. 1C and 1G). As had been observed previously (11), the peak V3 titers in some individuals (e.g. C11, C10, C17) appeared to decline with each successive immunization (FIGS. 1D, 1E, and 1F). After HIV-1 infection, two patterns of V3 reactivity were observed. Three subjects (C6, C7, and C10) showed large increases in titer to V3 domain peptides (FIGS. 1A, 1C, and 1E) whereas C8 (FIG. 1B) showed a large decrease in V3 titer. At the time of analysis, the data were insufficient to draw any conclusions regarding the changes in V3 titers in response to HIV-1 infection in subjects C11, C15 and C17.
The results obtained indicate that the ability to form antibodies reactive with the V3 domain at various time-points prior to HIV-1 infection is not a valid correlate of protective immunity against all strains of HIV-1.
CD4 Inhibition titers. Antibodies that block the binding of gp120 to CD4 represent a heterogeneous class of virus neutralizing antibodies. Some are known to bind to the C4 domain of gp120 [Nakamura et al.; J. Virol. 67:6179–91 (1993); Anderson et al.; J. Infect. Dis. 160:960–9 ((1989)], and some are known to recognize conformation dependent discontinuous epitopes [Berman et al.; J. Virol. 7:4464–9 (1992); Nakamura et al.; J. Virol. 67:6179–91 (1993); McKeating et al.; AIDS Research and Human Retroviruses 8:451–9 (1992); Ho et al.; J. Virol. 65:489–93 (1991); Barbas et al.; Proc. Natl. Acad. Sci. USA 91:3809–13 (1994)].
One way to detect antibodies to both types of epitopes is to measure the ability of vaccinee sera to prevent the binding of [125I]-labeled gp120 to cell surface CD4 [[Nakamura et al.; AIDS and Human Retroviruses 81875–85 (1992); Nakamura et al.; J. Virol. 67:6179–91 (1993)]. CD4 blocking titers were detected in all seven of the vaccinees prior to infection (FIG. 2) with peak titers that ranged from 1:10–1:300. At the last time point prior to infection, the CD4 titers in five of the seven vaccinees was low (1:30 or less). One vaccinee (C17), however, possessed a CD4 blocking titer of about 1:300 prior to infection (FIG. 2F). Thus, the lack of antibodies that block the binding of MN-rgp120 to CD4 cannot account for all of the infections. Large increases in CD4 blocking titers (1:100–1:1,000) were seen in five of the seven subjects after HIV-1 infection. These included vaccinees C6, C7, C8, C10, and C11. These results demonstrate that the CD4 blocking titers elicited by MN-rgp120 were lower than those elicited by natural infection.
Virus neutralizing activity. The virus neutralizing activity of antisera from MN-rgp120-immunized subjects was measured using a calorimetric assay that measured the viability of MT-4 cells after incubation with antibody treated virus (HIV-1MN). Since the actual date of infection was not known for any of the breakthrough infections, and serum samples were collected infrequently, the magnitude of the neutralizing antibody response at the time of infection is not known for any of the vaccinees.
Of the seven infections examined, the serum sample closest to the time of infection was that obtained from C7, where a neutralizing titer of 1:15 to HIV-1MN was present three weeks prior to detection of HIV-1 infection (Table 4). In all other cases, however, the interval between the last injection and the time of infection was 10 to 25 weeks.







TABLE 4







Neutralization Activity of Sera from Vaccinees


Infected with HIV-1














Week
C6
C8
C15
C7
C11
C10
C17

















0
 <10*
 <10*
 <10*
 <10*
 <10*
 <10*
 <10*


2
 <10
 <10
 <10
—
—
—
—


4
 <10*
 <10*
nd*
 <10*
 <10*
 <10*
 <10*


6
   10
   80
—
 <10
   30
  150
  150


8
—
—
nd*
—
—
—
—


10
—
—
   35
—
—
—
—


15
—
—
—
 <10
—
—
—


16
  150#
  250#
—
—
   30
   10
 <10


24


  150#
 <10*
   20*
 <10*
 <10*


26



   70
  500
  200
  400


30



—
—
   40
  100


33



   15
—
—
—


35



—
  100
—
—


36



   30#
—
   10
   40


52




   30*
 <10
 <10


54




  250
—
—


57




  100
—
—


63




   90
—
—


64




—
—
 <10


77




   40#
—
—


78





  500#
   10*


80






  100


84






   60


90






  150


104






  150#





*indicates immunization.


#indicates HIV-1 positive.


nd indicates not done.


— indicates sample not available.






When sera from the two early infections were examined (Table 4), one individual (C6) had a peak neutralizing titer of 1:10 ten weeks prior to detection of HIV-1 infection, whereas the other individual (C8) had a neutralizing titer of 1:80 ten weeks prior to detection of HIV-1 infection. Subject C15, who was immunized according to an accelerated immunization schedule, developed a neutralizing titer of 1:35 after the third injection, 14 weeks prior to HIV-1 infection. Subject C10, who had a peak neutralizing titer of 1:200 following the third immunization (week 24), had no detectable titer at week 52, six months prior to the first indication of HIV-1 infection (week 78).
Subject C11 possessed a neutralizing titer of 1:90 at fourteen weeks prior to detection of HIV-1 and a peak titer of 1:500 following the third immunization. Similarly vaccinee C17 had a neutralizing titer of 1:150 fourteen weeks prior to infection and a peak titer of 1:400 at two weeks after the third immunization.
Based on the rate of decay of the gp120 response of approximately two months [Belshe et al.; JAMA 272(6):475–80 (1994)], as well as the observation that neutralizing titers of 1:150 decayed to 1:10 in 10 weeks in vaccinees C10 and C17, it appears that neutralizing titers in C8, C15, C11, and C17 could have declined to 1:10 or less in the intervals between the last pre-infection serum sample and the time of HIV-1 detection.
The results of these studies demonstrated that all vaccinees developed some level of virus-neutralizing antibodies at some time prior to HIV-1 infection, and that the magnitude of the neutralizing response was probably low at the time of infection. In general, the magnitude of the virus-neutralizing response observed in the individuals that became infected with HIV-1 was comparable to that seen in non-infected vaccinees as described in Belshe et al.; JAMA 272(6):475–80 (1994).
Sequences of Viruses. To evaluate the similarity of the breakthrough viruses with the vaccine antigen, nucleotide sequences for gp120 from all seven breakthrough viruses were determined. Envelope glycoprotein genes were amplified from proviral DNA using the polymerase chain reaction. Sequences were obtained by direct amplification of DNA from lysates of gradient-purified lymphocytes obtained directly from patient blood without any intermediate tissue culture or amplification step.
A listing of the complete gp120 sequences (two clones per specimen) is provided in FIG. 3. All seven envelope glycoproteins possessed sequences typical of subtype (lade) B viruses. The overall homology with MN-rgp120 ranged from 69–80% (Table 5).







TABLE 5







Comparison of MN-rgp120 Sequence with Sequences


from Infected Vaccinees*
















MN
C6.1
C8.3
C7.2
C11.5
C10.5
C17.1
C15.2


















MN
100
79
78
70
75
69
80
72


C6.1

100
78
70
81
75
90
79


C8.3


100
68
80
76
84
83


C7.2



100
80
73
76
73


C11.5




100
75
70
80


C10.5





100
70
72


C17.1






100
87


C15.2







100





*Data indicate percent identity.






Interestingly, a high percentage (four of seven) of the breakthrough viruses differed from MN-rgp120 by 25–30% [Myers et al.; Retroviruses and AIDS Database, Los Alamos National Laboratory (1992 and 1995)]. Historically this degree of sequence variation is typical of inter-subtype (intra-clade) variation rather than intra-subtype variation which is expected to be in the 10–20% range [Myers et al.; Retroviruses and AIDS Database, Los Alamos National Laboratory (1992 and 19950]. Of the viruses with the greatest homology to MN-rgp120, two (C6 and C8) occurred as early infections, prior to complete immunization, and one (C17) occurred as a late infection.

Polymorphism in the V3 Domain. Of particular interest were polymorphisms in regions known to contain epitopes recognized by virus neutralizing antibodies. The best characterized neutralizing epitope, the principal neutralizing determinant (PND), occurs at the tip of the V3 loop. In subtype B viruses, approximately 60% possess the MN serotype-defining signature sequence, IGPGRAF (SEQ. ID. NO: 52), based on identity with the prototypic MN strain of HIV-1 [Berman et al.; Virol. 7:4464-9 (1992); Myers et al.; Retroviruses and AIDS Database, Los Alamos National Laboratory (1992 and 1995); La Rosa et al.; Science 249:932–5 (1990)].
Three of the viruses (C6, C8, and C17) possessed the MN serotype signature sequence (FIG. 3). In contrast, four viruses possessed sequences with radical amino acid substitutions in the PND [IGPGRAW (7), LGPGSTF (11), IGPGRVL (10), and IGPGSAF (15)] (SEQ. ID. NOs. 53–56), respectively), and therefore were classified as “non-MN like” viruses. Of note, each of the four “non-MN-like” sequences were rare (Table 6) and were not typical of the most common “non-MN” variants of subtype B viruses [Myers et al.; Retroviruses and AIDS Database, Los Alamos National Laboratory (1992 and 1995)].







TABLE 6







Frequency of Polymorphisms at the Principal


Neutralizing Determinant in HIV-1 Infected


Individuals Immunized with MN-rgp120*










Ob-




served
Dataset Frequency












V3 Sequence
Fre-
GNE
LANL
LANL.1
LaRosa













Sequence
n
quency
(n = 52)
(n = 519)
(n = 160)
(n = 245)
















GPGRAF
3
0.42
0.67
0.57
0.66
0.60


GPGRAW
1
0.14
0.03
0.013
0.06
0.010


GPGRVL
1
0.14
<0.02
0.004
<0.006
<0.008


GPGSTF**
1
0.14
<0.02
<0.002
<0.006
<0.004


GPGSAF
1
0.14
0.02
0.011
<0.006
<0.004





*Data set GNE refers to a collection of 52 independent isolates collected in 1992; dataset LANL refers to a collection of 519 sequences reported by Myers et al., Retroviruses and AIDS Database, Los Alamos National Laboratory 1992 and 1995; LANL.1 refers to a collection of 160 epidemiologically unlinked individuals provided by B. Korber (personal communication) ; dataset La Rosa refers to sequence data reported by La Rosa et al., Science 249:932-5 (1990) .


**Sequences were not present in the data sets examined.






The prevalence of viruses with PND sequences matching the breakthrough viruses ranged from a high of 1.3% (C7) to a low of 0.2% (C11) in a listing of 519 subtype B sequences compiled by the Los Alamos National Laboratory [Myers et al.; Retroviruses and AIDS Database, Los Alamos National Laboratory (1992 and 1995)]. Similarly low frequencies were observed in three other independently derived data sets (Table 6). The occurrence of these sequences did not differ significantly between data sets collected prior to 1985 [La Rosa et al.; Science 249:932–5 (1990)] and data collected 1992, or from a set of 160 epidemiologically unlinked individuals (B. Korber, personal communication). All four sets of data agreed that the prevalence of viruses with MN-like PND sequences was in the range of 60%. Based on this data, four of the seven breakthrough infections were determined to be caused by viruses that fell outside of the spectrum of viruses that the vaccine was expected to prevent.
Other features of breakthrough virus V3 domains. Like MN-rgp120, the V3 domains of all of the breakthrough viruses were 36 amino acids in length. However, all seven viruses differed from MN-rgp120 with respect to the number of glycosylation sites and with respect to the syncytium-inducing (SI) signature sequence.
The sequence of MN-rgp120 is somewhat unusual [Myers et al.; Retroviruses and AIDS Database, Los Alamos National Laboratory (1992 and 1995)] in that it lacks an N-linked glycosylation site at position 306 in the V3 domain. The lack of this glycosylation site does not appear to be antigenically significant since antisera to MN-rgp120 are known to neutralize a variety of viruses (e.g. SF-2, DU6587-5, DU4489-5, CC) that possess a glycosylation site at this position [Berman et al.; J. Virol. 7:4464–9 (1992)]
In addition, the V3 domain of MN-rgp120 possessed sequence polymorphisms (R at position 311, K at position 324, K at position 328) typical of syncytium inducing viruses [Fouchier et al.; J. Virol. 66:3183–87 (1992)], whereas all seven breakthrough viruses possessed sequences associated with non-syncytium-inducing viruses. Syncytium-inducing viruses have been associated with rapid disease progression [Tersmette et al.; J. Virol. 62:2026–32 (1988)] and T cell tropism [O'Brien et al.; Nature (London) 348:69–73 (1990); Shioda et al.; Nature (London) 349:167–9 (1991)]. To date viruses with these properties have not been recovered from any of the MN-rgp120 immunized volunteers.
Polymorphism in the V1, V2 and C4 domains. Previous investigations have identified additional neutralizing epitopes in the V1, V2 and C4 domains of gp120 [Nakamura et al.; J. Virol. 67:6179–91 (1993); McKeating et al.; AIDS Research and Human Retroviruses 8:451–9 (1992); Ho et al.; J. Virol. 65:489–93 (1991); Barbas et al.; Proc. Natl. Acad. Sci. USA 91:3809–13 (1994); McKeating et al.; J. Virol. 67:4932–44 (1993); Moore et al.; J. Virol. 67:6136–6151 (1993); Davis et al.; J. Gen. Virol. 74:2609–17 (1993)].
The best characterized of these neutralizing epitopes is in the C4 domain which has attracted special attention because antibodies binding to this area are known to block the binding of gp120 to CD4 [Moore et al.; AIDS 3:155–63 (1989); McKeating et al.; AIDS Research and Human Retroviruses 8:451–9 (1992)]. Because the epitope is located in a conserved (C) domain, naturally-occurring polymorphism in this region is far more limited than in other neutralizing epitopes. Nakamura et al.; J. Virol. 67:6179–91 (1993) reported that the binding of a number of neutralizing MAbs was dependent on K at position 429.
Comparison of the sequence of MN-rgp120 with other strains of HIV-1 showed that a common polymorphism, involving the substitution of E for K, occurs at this position. Indeed, substrains of the same virus isolate often show polymorphism at this position. The HXB2 substrain of HIV-1LAI contains K at position 429, whereas the BH10, IIIB, and LAV substrains of the HIV-1LAI contain E at this position [Nakamura et al.; J. Virol. 67:6179–91 (1993)]. Similarly, the 1984 isolate of HIV-1MN exhibited E at this position, while the 1990 isolate of HIV-1MN, used to produce MN-rgp120, possessed K at this position.
When the sequences of the infected vaccine recipients were examined (FIG. 3), the virus from subject C17, like MN-rgp120 contained K at position 429, whereas the six other viruses that differed from the vaccine immunogen possessed E at this position. These results demonstrated that six of the seven breakthrough viruses differed from the vaccine immunogen at the CD4-blocking, neutralizing epitope in the C4 domain of gp120.
Studies with monoclonal antibodies have defined neutralizing epitopes in the V1 and V2 domains of gp120 [McKeating et al.; J. Virol. 67:4932–44 (1993); Moore et al.; J. Virol. 67:6136–6151 (1993); Davis et al.; J. Gen. Virol. 74:2609–17 (1993)]. Like the polymorphisms that occur in the C4 domain, the V2 domains exhibit several common polymorphisms that affect the binding of virus neutralizing antibodies. One such polymorphism occurs at position 171 which is critically important for the binding of murine MAb 1025, whereas residue 187 is important for the binding of MAb several MAbs represented by 1088.
When the V2 domain sequences were examined (FIG. 3), all of the infected-vaccinee viruses differed from MN-rgp120 in that R replaced G at position 171 and I or V replaced E at position 187. Antibodies recognizing these adjacent sites in the V2 domain of MN-rgp120 would not be expected to neutralize viruses with radical amino acid substitutions at these position. Thus, all seven breakthrough viruses differed from MN-rgp120 at a neutralizing epitope in the V2 domain of gp120.
Other neutralizing epitopes have been reported in the V1 domain of gp120 [O'Brien et al.; Nature (London) 348:69–73 (1990); McKeating et al.; J. Virol. 67:4932–44 (1993)]. Although the neutralizing epitopes in the V1 domain of MN-rgp120 have not been characterized, the polymorphism seen among the breakthrough viruses in this region was interesting. Particularly striking (FIG. 3) was that the length of this domain ranged from 20 amino acids (C17) to 45 amino acids (C6), and the number of N-linked glycosylation sites ranged from 2 to 6. In contrast, the Vi domain of MN-rgp120 is 31 amino acids in length and encodes three N-linked glycosylation sites.
Although examination of sequence databases suggest that variation in the V2 region is comparable to the V1 region, the V2 region of the breakthrough viruses showed less variation than expected. Specifically, the length of the V2 region ranged from 36 amino acids (C7) to 39 amino acids in length, with six of seven viruses containing three N-linked glycosylation sites in this domain. A high degree of polymorphism was found in the V4 region where sequences ranged from 26 (C10) to 33 (C15, C7) amino acids in length and contained either 4 or 5 N-linked glycosylation sites.
Antigenicity of envelope glycoproteins from breakthrough viruses. To determine the significance of sequence variation on glycoprotein antigenicity, recombinant gp120 was prepared from the viruses of all seven infected vaccinees (FIG. 4). In these studies a series of MAbs was assembled and their binding to MN-rgp120 was compared to that of rgp120 from the vaccinee isolates by ELISA (Table 7).







TABLE 7







Relative Reactivity* of MAb Binding to rgp120 from


Infected Subjects Compared with Binding to MN-rgp120












V3
Discontinuous
C8
V2













gp120
1034
50.1
1.5E
1025
1024
1088
















MN
1.0
1.00
1.00
1.00
1.00
1.00


C6.1
0.37
0.37
0.17
0.00
0.00
0.00


C6.5
0.33
0.33
0.75
0.00
0.00
0.00


C8.3
0.11
0.37
0.38
0.00
0.00
0.00


C8.6
0.14
0.34
0.29
0.00
0.00
0.00


C7.2
0.47
0.60
0.71
0.00
0.00
0.00


C11.5
0.00
0.00
0.17
0.00
0.00
0.00


C11.7
0.00
0.00
0.17
0.00
0.00
0.00


C10.5
0.33
0.40
0.46
0.24
0.03
0.04


C10.7
0.42
0.48
0.50
0.29
0.07
0.09


C17.1
0.33
0.52
0.33
0.00
0.30
0.07


C17.3
0.37
0.56
0.33
0.00
0.38
0.06


C15.2
0.00
0.47
0.92
0.00
0.00
0.00


C15.3
0.00
0.37
0.63
0.00
0.00
0.00





*Relative reactivity values represent ratio of optical densities obtained with rgp120 from patient isolates divided by optical density obtained for MN-rgp120 at a MAb concentration of 2 micrograms per milliliter.






In control experiments, the binding of MAb 5B6 (which is specific for the HSV gD-1 flag epitope fused to the N terminus of all of the rgp120 protein) was used to standardize the amount of gp120 from each isolate (FIG. 5A). These studies demonstrated that the assay was carried out under conditions where equivalent amount of rgp120s were captured onto wells of microtiter plates.
The antigenic structure of the V3 domain was examined using the 1034 MAb (isolated from mice immunized with MN-rgp120 as described in Nakamura et al.; J. Virol. 67:6179–91 (1993) and the 50.1 MAb (prepared from mice immunized with a synthetic V3 domain peptide as described in Rini et al.; Proc. Nati. Acad. Sd. USA 90:6325–9 (1993). Both MAbs are known to exhibit potent virus neutralizing activity. When binding to the recombinant proteins was examined, the MAb binding to MN-rgp120 was at least 10-fold greater than to any of the breakthrough virus envelope proteins (FIGS. 5B and C). Surprisingly, rgp120 from the three patient isolates (C8, C6, and C17) that possessed the MN serotype-defining sequence, IGPGRAF (SEQ. ID. NO: 52), varied from one another in their MAb binding activity. Thus, the binding of MAb 1034 and MAb 50.1 to rgp120 from C17 was significantly greater than the binding to rgp20s from C6 and C8.
A distinction in the epitopes recognized by these MAbs was evident since C6-rgp120 and C8-rgp120 gave comparable binding with 50.1, whereas 1034 bound better to the C6-derived protein than the C8-derived protein. The poorest MAb reactivity was with rgpl20s from C11 and C15. This result was consistent with sequence analysis demonstrating that these two viruses both possessed the radical substitution of S for R at position 18 in the V3 domain. Surprisingly, both of these MAbs exhibited better than expected binding to rgp120 from the C7 and C10 viruses. Like MN-rgp120, both proteins contained the penta-peptide, IGPGR sequence (SEQ. ID. NO: 57) in the V3 loop, but differed from MN-rgp120 in that V and L replaced A and F at positions 319 and 320 in gp120 from C10, and W replaced F at position 320 in gp120 from C7. These results indicate that R at position 318 is essential for the binding of these two MAbs, and that the epitopes recognized by 1034 and 50.1 are not completely destroyed by the hydrophobic substitutions at positions 319 and 320.
As predicted from the sequence data, there was little if any binding to the breakthrough virus rgp120s using MAbs (1088 and 1025) directed to the V2 region of MN-rgp120 Also consistent with sequence data was the observation that MAb 1024 directed to the C4 domain of MN-rgp120 gave some reactivity with C17-rgp120 which, like MN-rgp120 contained K at position 429, but gave no reactivity with the other isolates that contained E at residue 429.
Together, these studies demonstrated that the antigenic structure of all seven breakthrough viruses differed from the vaccine immunogen at three well characterized neutralizing epitopes.
A totally different pattern of reactivity was observed with the human hybridoma, MAb 15e, prepared from an HIV-1 infected individual as described in Ho et al.; J. Virol. 65:489–93 (1991). With this MAb, the greatest binding was achieved with MN-rgp120 and rgp120 from C7, and the poorest reactivity was seen with the two clones of rgp120 from the C11. Moderate, but comparable reactivity was seen with rgp120s from the C10 and C17.
These results demonstrate that the 15e epitope is polymorphic, and that the epitope is conserved on MN-rgp120 and rgp120 from C7, but has been lost on rgp120s from C11. Interestingly, the two different clones of gp120 derived from C6 gave strikingly different patterns of antibody binding. Thus, rgp120 from clone C6.5 exhibited strong reactivity with this antibody, whereas rgp120 from clones C6.1 exhibited significantly weaker activity with this MAb. Comparison of sequence data (FIG. 3) showed that the two C6 clones differed at 6 amino acid positions. Based on comparative binding to the other viral proteins of known sequence, it appeared that the substitution of K for I at position 351 in the C3 domain of gp120 could account for the difference in binding activity. This result is also consistent with both clones of C11 similarly containing a positively-charged K at this position, and also being poorly reactive with this MAb. Alternatively, a T for I substitution at position 439 in the C4 domain could account for the difference in 15e binding between C6.1 and C6.5. Although the inability of the two C11 clones to bind 15e cannot be explained by polymorphism at this position in the C4 domain, they could be affected by the adjacent T for M substitution at position 434.
Discussion
In these studies, the viruses and immune responses in seven of nine vaccinees who became infected with HIV-1 vaccine through high risk activity while participating in Phase I or Phase 2 trials of MN-rgp120, a candidate HIV-1 vaccine were analyzed. Such infections would be expected to occur for one of two reasons: 1) lack of sufficient immune response at the time of infection; or 2) infection with viruses that fall outiside of the antigenic spectrum expected to be covered by the vaccine immunogen. The data indicate that both explanations may be involved with the infections observed (Table 8).







TABLE 8







Summary of Breakthrough Infections










MN-rgp120
Homoloqous to MN-rgp120













Adequate
Homology
V3
C4
V2


Case No.
Immunization
(%)
PND
Epitope
Epitope





C6
−
79
+
−
−


C8
−
78
+
−
−


C15
−
72
−
−
−


C7
−
70
−
−
−


C11
+
75
−
−
−


C10
+
69
−
−
−


C17
+
80
+
+
−









Two of the infections occurred in individuals who failed to receive the minimum three doses of vaccine typically required for the induction of protective immunity with protein subunit vaccines (e.g. hepatitis B virus formulated in alum adjuvant as described in Francis et al.; Ann. Int. Med. 97:362–6 (1982). Two additional breakthrough infections occurred in vaccinees who had weak or undetectable primary (C7) and booster (C15) responses. Of the three individuals who became infected with HIV-1 after receiving three or more productive immunizations (C10, C11, and C17), at least two, and possibly all three, appear to have become infected more than six months after receiving their last immunization. Because antibody titers to MN-rgp120 typically decay with a half-time of 2 to 2.5 months [Belshe et al.; JAMA 272(6):475–80 (1994); Berman et al.; AIDS 8:591–601 (1994)], antibody titers would be expected to have decayed at least eight-fold and possibly as much as sixty four-fold at the time of infection. Thus, the lack of a sufficient immune response at the time of infection represents a potential explanation for at least six of the seven breakthrough infections.
Data from vaccine efficacy studies in gp160 immunized chimpanzees [McElrath et al.; Longitudinal Vaccine-Induced Immunity and Risk Behavior of Study Participants in AVEG Phase II Protocol 201. In: Abstracts from Eighth Annual Meeting of the National Cooperative Vaccine Development Groups for AIDS. Bethseda, Md. 1996:216] challenged with HIV-1, and gp120-immunized rhesus macaques challenged with a chimeric SIV/HIV-1 virus (SHIV) suggest that the magnitude of the neutralizing antibody response at the time of infection is a critical correlate of protective immunity. If maintaining neutralizing antibody titers proves to be a valid correlate of protective immunity in humans, then formulations (e.g. novel adjuvants) or immunization regimes (frequent boosting) designed to maximize the antibody responses may be required to achieve long lasting protection. Use of a booster every six months may be advantageous.
The other likely explanation for the late infections is the antigenic difference between the vaccine and the breakthrough virus envelope glycoproteins. This explanation is supported by the observation that four of the seven breakthrough viruses possessed envelope glycoproteins that differed from the MN-rgp120 by 25–30% at the amino acid level. Differences of this magnitude have historically [Myers et al.; Retroviruses and AIDS Database, Los Alamos National Laboratory (1992 and 1995)] been associated with inter-subtype variation and far exceeds the average 10–20% variation expected for viruses within the same subtype.
Although the biologic significance of sequence variation in many regions of the envelope glycoprotein is unclear, polymorphism at neutralizing epitopes is an important factor that affects vaccine efficacy. Previous studies [Salmon-Ceron et al.; AIDS Res. and Human Retroviruses 11:1479–86 (1996); Javaherian et al.; Science 250:1590—3 (1990)] have demonstrated that the breadth of neutralizing activity that could be elicited by HIV-1 envelope derived vaccines was critically dependent on the sequence of epitopes in the V3 domain (e.g.; the PND). Thus, candidate vaccines based on the LAI strain of HIV-1 (the prototypic “non-MN-like” subtype B virus), exhibited little or no cross neutralizing activity with subtype B viruses, whereas vaccines that contained the “MN-like-” PND sequence (IGPGRAF) (SEQ. ID. NO: 52) exhibited broad cross neutralizing activity. That four of the seven breakthrough viruses possessed envelope glycoproteins with radical amino acid substitutions in the PND is consistent with the explanation that differences in antigenic structure explain some of these infections.
Over the last few years, it has become clear that polymorphism among “MN-like” viruses occurs at neutralizing epitopes outside of the PND. The best example occurs in the C4 domain where two antigenically distinct variants are distinguished by the presence of either K or E at position 429 [Moore et al.; AIDS 3:155–63 (1989)]. Because six of the seven breakthrough viruses differed from the vaccine strain in that they contained E rather than K at position 429, antibodies raised to the C4 domain of MN-rgp120 were unlikely to neutralize the viruses infecting in six of the seven vaccinees.
Other neutralizing epitopes are known to be present in the V1 and V2 domains of gp120. Although these regions are highly variable, due to insertions and deletions, neutralizing epitopes have been described by McKeating et al.; J. Virol. 67:4932–44 (1993); Moore et al.; J. Virol. 67:6136–6151 (1993); and Davis et al.; J. Gen. Virol. 74:2609–17 (1993). Several of these epitopes overlap an amino terminal sequence of the V2 domain containing the tri-peptide sequence RDK at positions corresponding to 142 to 144 of MN-rgp120 [McKeating et al.; J. Virol. 67:4932–44 (1993); Moore et al.; J. Virol. 67:6136–6151 (1993)]. Like the C4 epitope, variation in this sequence is known to occur between different substrains derived from the same parental isolate. Since all seven breakthrough viruses differed from MN-rgp120 in that they possessed the RDK sequence, rather than the GDK sequence present in the vaccine antigen, neutralizing antibodies to the V2 domain of MN-rgp120 would not have been expected neutralize any of the viruses recovered from the vaccinees immunized with MN-rgp120.
Although polymorphisms at neutralizing epitopes might account for the lack of protection in most of the infections, this does not appear to explain the infection of vaccinee C17, who was infected by a virus that matched MN-rgp120 in the V3 and C4 domains. If a difference in sequence was responsible for the lack of protection in this case, the critical difference might relate to the unusual sequence in the V1 domain of gp120 from this breakthrough virus. Several studies have shown that the V1 domain possesses epitopes recognized by virus neutralizing monoclonal antibodies [McKeating et al.; J. Virol. 67:4932–44 (1993); Davis et al.; J. Gen. Virol. 74:2609–17 (1993); Kayman et al.; J. Virol. 68:400–410 (1994)].
Although far less is known about the V1 epitopes relative to other neutralizing sites, the V1 epitopes appear to be conformation-dependent, and antisera from HIV-1 infected individuals recognize epitopes in the V1 and V2 domains [McKeating et al.; J. Virol. 67:4932–44 (1993); Kayman et al.; J. Virol. 68:400–410 (1994)]. The V1 sequence of the virus from C17 is noteworthy because it is smaller and contains fewer N-linked glycosylation sites than that of MN-rgp120 or any of the other breakthrough viruses. By the same token, the envelope glycoproteins from C11 and C6 are noteworthy because they are significantly larger and contain more glycosylation sites than MN-rgp120 or the other breakthrough viruses.
While differences in amino acid sequence can provide clues to differences in antigenic structure, the consequences of such polymorphism can only be proven through antibody binding studies. To correlate differences in sequence with differences in antigenic structure, gp120 from two clones each of all seven breakthrough viruses was expressed and the antigenicity of the clones with a panel of monoclonal antibodies was examined. As predicted from the sequence data, none of the breakthrough virus envelope glycoproteins reacted with neutralizing MAbs to the V2 domain of MN-rgp120. When MAbs to the C4 domain were examined, only the C17 envelope glycoprotein (that matched MN-rgp120 with respect to K429) showed significant, albeit lower, binding. Surprisingly, the three breakthrough envelope glycoproteins that contained the subtype B PND consensus sequence, IGPGRAF (SEQ. ID. NO: 52), gave poor reactivity with all three PND directed MAbs, even though they possessed PND sequences closely related to the vaccine immunogen. Thus, all three of the vaccinee isolates appeared to possess changes outside of the recognition site that interfered with MAb binding.
It has been known for many years that resistance to neutralization in vitro can sometimes be attributed to mutations in remote sequences that alter the conformation of neutralizing epitopes and interfere with recognition by virus neutralizing antibodies [Nara et al.; J. Virol. 64:3779–91 (1990); Cordonnier et al.; Nature 340:571–4 (1989)]. Together, these results indicate that the antigenic structure of the envelope glycoproteins recovered from the breakthrough viruses differed significantly from that of the vaccine antigen.
A novel result was the localization of residues in the C3 domain that appeared to affect the binding of the virus neutralizing human MAb, 15e. This MAb is known to recognize a discontinuous epitope, block CD4 binding, and neutralize a variety of laboratory and primary isolates of HIV-1 [Ho et al.; J. Virol. 65:489–93 (1991); Thali et al.; J. Virol. 66:5635–5641 (1992); Moore et al.; AIDS Res. Hum. Retroviruses 9:1179–1187 (1993)].
Comparative binding to envelope glycoproteins from the breakthrough viruses indicated that recognition by this antibody is critically dependent on residues in the C3 or C4 domains of gp120. The unique occurrence of a positively charged K at position 351 in the C3 domain provides a common explanation for the inability of the C11.5, C11.7 and C6.1 strains of HIV-1 to bind to 15e. Alternatively, it is possible that different amino acid substitutions in different locations account for the failure of 15e to bind to rgp120s from the C6 and C11 clones. The only obvious positions where substitutions of this type occur are in the C4 domain where T replaces M at 434 (C11) and T replaces I at 439.
The present studies demonstrate that the current formulation of MN-rgp120 is less than 100% effective against HIV-1 infection. Based on previous in vitro and in vivo studies with MN-rgp120, protection from natural HIV-1 infection in humans is expected to depend on a threshold concentration of virus-neutralizing antibodies, and antigenic similarity between the vaccine immunogen and the challenge virus.
In this regard, only one of the seven breakthrough infections (C17) was unexpected. This individual received a full course of immunizations yet became infected with a virus similar to MN-rgp120 at least two important neutralizing epitopes (V3 and C4 domains). This infection might be related to the magnitude of the antibody response at the time of infection, or antigenic differences between the breakthrough virus and the vaccine strain, or circumstances of infection (e.g., ulcerative lesions, infection by donor with acute infection or high viremia), not monitored in this protocol. Alternatively this individual may represent a true vaccine failure, without clear explanation.
On balance, the analysis of breakthrough infections described herein did not uncover any data that would discourage the continued development of MN-rgp120 as a vaccine to prevent HIV-1 infection. The results support speculation that enhancing vaccine immunogenicity (as by additional booster immunizations) may be required to maintain long term protective immunity, and that the addition of rgp120 from other antigenically different strains of virus in addition to MN-rgp120 are useful to expand the breadth of protection.
The availability of viruses and viral glycoproteins derived from breakthrough infections may provide an important means to streamline the process of identifying new antigens for inclusion into a multivalent vaccine. Recombinant viral glycoproteins prepared from breakthrough viruses, by definition, possess antigenic structures that are significantly different from MN-rgp120, and are be representative of viruses currently being transmitted. Thus, combining rgp120 from breakthrough viruses with MN-rgp120 is an effective way complement and significantly expand antigenic complexity and increase breadth of cross neutralizing activity.

## Claims

1. A composition comprising one or more oligonucleotides capable of expressing a first polypeptide comprising a gp120 MN sequence as identified by Sequence ID No. 41, or a fragment thereof, and a polypeptide comprising a breakthrough isolate gp120 sequence selected from the group consisting of Sequence ID Nos. 2, 5, 8, 10, 12, 16, 19, 23, 25, 28, 31, 33, 36, 39, and fragments thereof, in a suitable carrier, wherein each of said fragments comprises at least the V2, V3, and C4 domains of gp120.

2. The composition of claim 1 wherein a single oligonucleotide expresses the first polypeptide and the polypeptide comprising a breakthrough isolate gp120 sequence or a fragment thereof.

3. The composition of claim 1 wherein said oligonucleotide or at least one of said oligonucleotides is a DNA molecule.

4. The composition of claim 1 wherein said oligonucleotide or at least one of said oligonucleotides is a viral vector.

5. A composition comprising:
a) an oligonucleotide capable of expressing a first polypeptide comprising a first gp120 sequence or a fragment thereof; and
b) an oligonucleotide capable of expressing a polypeptide comprising a breakthrough isolate gp120 sequence, or a fragment thereof, wherein said breakthrough isolate gp120 sequence is selected from the group consisting of Sequence ID Nos. 2, 5, 8, 10, 12, 16, 19, 23, 25, 28, 31, 33, 36, and 39;
wherein each of said fragments comprises at least the V2, V3, and C4 domains of gp120, and said oligonucleotides are in a suitable carrier.

6. The composition of claim 5 wherein a single oligonucleotide expresses the first polypeptide and the polypeptide comprising a breakthrough isolate gp120 sequence or a fragment thereof.

7. The composition of claim 5 wherein said first gp120 sequence comprises gp120 MN as identified by Sequence ID No. 41, gp120 CM244, gp120 MN-GNE6 which comprises Sequence ID Nos. 43 and 44, or gp120 MN-GNE8 as identified by Sequence ID No. 46.

8. The composition of claim 5 wherein said composition additionally comprises an oligonucleotide capable of expressing a second polypeptide comprising a second gp120 sequence comprising gp120 MN as identified by Sequence ID No. 41, gp120 CM244, gp120 MN-GNE6 which comprises Sequence ID Nos. 43 and 44, gp120 MN-GNE8 as identified by Sequence ID No. 46, or a fragment thereof, wherein said second gp120 sequence is different from said first gp120 sequence.

9. The composition of claim 8 wherein said first gp120 sequence comprises gp120 MN as identified by Sequence ID No. 41 and said second gp120 sequence comprises gp120 CM244.

10. The composition of claim 8 wherein said first gp120 sequence comprises gp120 MN as identified by Sequence ID No. 41 and said second gp120 sequence comprises gp120 MN-GNE8 as identified by Sequence ID No. 46.

11. The composition of claim 8 wherein said breakthrough isolate gp120 sequence or fragment is from a breakthrough isolate obtained from an individual immunized with said first and second polypeptides.

12. The composition of claim 5 wherein at least one of said oligonucleotides is a DNA molecule.

13. The composition of claim 5 wherein at least one of said oligonucleotides is a viral vector.

14. A method for making a composition comprising:
a) providing a first composition comprising an oligonucleotide capable of expressing a first polypeptide comprising a first gp120 sequence or a fragment thereof;
b) obtaining a breakthrough isolate from an individual immunized with said first polypeptide;
c) selecting a breakthrough isolate gp120 sequence, or a fragment thereof, from said breakthrough isolate; and
d) adding an oligonucleotide capable of expressing a polypeptide comprising said breakthrough isolate gp120 sequence, or fragment thereof, to said first composition;
wherein each of said fragments comprises at least the V2, V3, and C4 domains of gp120.

15. The method of claim 14 wherein said first gp120 sequence is from a macrophage-tropic HIV-1 strain.

16. The method of claim 14 wherein said first gp120 sequence is from a T-cell-tropic HIV-1 strain.

17. The method of claim 14 wherein said first composition additionally comprises an oligonucleotide capable of expressing a second polypeptide comprising a second gp120 sequence, or a fragment thereof, from a macrophage-tropic HIV-1 strain.

18. The method of claim 17 wherein said first and second gp120 sequences bind to different chemokine receptors.

19. The method of claim 18 wherein said first gp120 sequence binds to CC-CKR-5, and said second gp120 sequence binds to CXC-CKR-4.

20. The method of claim 14 where said composition additionally comprises a virus engineered to induce a cytotoxic T-cell response.

21. The method of claim 14 wherein said oligonucleotides are DNA molecules.

22. The method of claim 14 wherein said oligonucleotides are viral vectors.