604
CONCISE COMMUNICATIONS
Molecular Epidemiology of Envelope Glycoprotein H of Human Cytomegalovirus SUDweD Chou
Medical and Research Services. VA Medical Center. and Division of Infectious Diseases. Oregon Health Sciences University. Portland. Oregon
Envelope glycoproteins Band H (gB, gH) ofcytomegalovirus (CMV) are probably essential for viral entry into host cells and are important targets of human immune responses such as neutralizing antibody [I]. The major glycoprotein (gB) has been studied extensively and shown to contain epitopes in both its gp5 5 and gp I 16 components that are recognized by murine monoclonal and human neutralizing antibody [2, 3]. Sequence data indicate that interstrain variation in gB is strongly clustered at several distinct loci and that clinical strains adopt one of up to four group-specific peptide configurations at these loci [4, 5]. gH (gp86) was initially characterized by sequencing and reactivity with a neutralizing murine monoclonal antibody [6]. In its glycosylated form, it has a size of 86 kDa and appears to bind a 92.5-kDa cellular receptor [7]. Neutralizing human monoclonal antibodies have been produced to gH and have been entered into clinical trials [8]. The published AD 169 and Towne strain gH sequences show significant variation in the first 37 codons and scattered changes in the rest of the molecule [6]. The purpose of the present study was to determine the diversity of gH sequences among clinical strains as an indication of the probable cross-reactivity of immune responses directed to this molecule and to aid in the design of diagnostic primers and amplification targets.
Received 3 February 1992; revised 22 April 1992. Financial support: Department of Veterans Affairs research funds. Ten complete gH coding sequences have been submitted to Genbank, and the accession and strain numbers. respectively. are as follows: M94228. C074; M94229. C076; M94230. C079; M94231. C325; M94232. C327; M94233. C336; M94234. C338; M94235. C351; M94236. C352; M94237. C353. Reprints or correspondence: Dr. Sunwen Chou. Infectious Disease Section II IF. VA Medical Center. P.O. Box 1034. Portland OR 97207. The Journal of Infectious Diseases 1992;166:604-7 © 1992 by The University of Chicago. All rights reserved. 0022-1899/92/6603-0022$01.00
Methods Ten clinical CMV strains were selected for sequencing from a library of > 30 strains that had been screened for diversity by restriction analysis of various target sequences amplified bythe polymerase chain reaction (PCR) [9]. They were genetically distinct and obtained from epidemiologically unrelated transplant recipients. Clinical isolates appearing to contain multiple strains by restriction analysis were excluded. To avoid artifactual sequence change during passage, plaque purification was not attempted except for strains C325 and C327, which were plaquepurified directly from the urine specimen [10]. These plaque-purified strains were sequenced at passage 11; DNA from all other strains was extracted within 6 passagesof primary isolation. Complete gH coding sequences were determined by dideoxy chain termination reactions on PCR-amplified biotinylated templates after attachment to streptavidin-coated magnetic beads as previously described for gB [5]. A separate PCR was done to generate a template for each sequencing reaction by using a biotinylated primer based on reference sequences at or beyond either end of the gH coding sequence (figure 1) paired with a primer on the complementary strand, which was located in a 5' direction with respect to the intended sequencing primer. Overlapping coverage of the 2.2-kb coding sequence was achieved by using synthetic oligonucleotide primers of conserved sequence (AD 169 and Towne strains), spaced :::;300 basesapart and representing both DNA strands of the region sequenced (figure 1). Thus, numerous separate PCR amplifications, with a variety of primer pairs, were done on a given viral DNA extract to generate the complete gH coding sequence.
Results As was found with gB [5], sequencing of PCR-generated templates provided coherent overlapping data for each strain despite the use of unpurified amplification products generated separately for each sequencing reaction. Thus, the strains sequenced here each appeared to contain a single ge-
Downloaded from http://jid.oxfordjournals.org/ at Michigan State University on June 18, 2015
The complete envelope glycoprotein H (gH) coding sequences of 10 clinical strains of cytomegalovirus (CMV) were determined and compared with those of laboratory strains AD169 and Towne. Their translated peptide sequences segregated into two groups, exemplified by AD169 and Towne. Peptide variation was mostly group-specific and was clustered in the first 37 amino acids, including the signal sequence; in the rest of the molecule, there were scattered amino acid substitutions, usually in single residues. Compared with CMV envelope glycoprotein B, gH is more highly conserved among strains and may be expected to have limited immunologic diversity.
Concise Communications
1ID 1992; 166 (September)
FigUre 1. CMV glycoprotein H amplification and sequencing primers.
605
Reverse (noncoding) strand
Name
Sequence (5'-3')
Name
Sequence (5'-3')
gH175 gH190 gH203* gH299 gH571 gHII08 gHl153 gH1321 gH1471 gH1672 gH1873
CTCCTTCTCTCGGGTGTAAC AGCAGCCTCCGTAACAGCAC CCACCTGGATCACGCCGCTG GTCCTCTGGCGGAGCAGTT CACACTTTAACCAGACCTGTAT CGCCAGGCCGCACTCTT CGACTTCAACTACCTGGACC CTACGACAGATCGCCGA GTAGAAACGGGCCTCTG ACCATGCAACCAAGCAC TGCGAACTAACGCGCAA
gH172 gH495 gH622 gH803 gH1288 gH1688 gH2002 gH2184 gH2228* gH2373 gH2424*
TGGTGTTTTCACGCAGGAA AGGTATTGACAGATCAATGG GTAGGTGTTAAGTCTCTG AAGTTGTCGCGTTGATAGGG TGCTAATGCGTAGGCGAAGG GTGCTTGGTTGCATGGT ATGTACATGCACGACTCGGA CGATAGCGCGTAGACGGACAT CATGTCTTGAGCATGCGGTA ACCATCACACCGTGTGGCGT ACCCATAACAGTACCTCGTA
*Biotinylated primers used to generate sequencing templatesC)
notype, whereas attempted sequencing of isolates known to contain mixtures of strains has resulted in conflicting data from different sequencing reactions (data not shown). Of the 743 codons in gH, 157 (21 %) showed interstrain variation at the nucleotide level and 34 (4.6%) at the peptide level. At both levels, but especially at the peptide level, the coding sequences segregated into two groups. AD 169 and related strains formed group 1, with a coding sequence of 743 codons, while Towne and related strains formed group 2, with a coding sequence of 742 codons. Strains in the same group had ~8 amino acid differences; strains in opposite groups had '" 30. The numbering system used here is from group 1; the group 2 strains all had a deletion at codon 36. Of the 10 clinical strains sequenced, 6 were in group 1 and 4 were in group 2. Restriction enzyme (HhaI) digestion of a PCR-amplified target (primers gH203jgH 172) yielded one of two fragment patterns that identified the gH group of a given strain; by this analysis, 20 of 40 additional random clinical strains from our hospital were in group 1 and the rest were in group 2. Thus, gH groups 1 and 2 were about equally common. Figure 2A shows the distribution of variable codons that affected peptide encoding. Nearly all of the clustered variation was in the first 37 codons, 21 of which are the probable signal sequence. The remaining variable codons were widely scattered and usually varied in a group-specific manner, although a few isolated mutations specific to a single strain were noted (figure 2A). More than half of the amino acid variation involved some change in polarity of the residue involved (figure 2B). There were six potential glycosylation sites (Asn ...Ser or Asn ...Thr) that were completely conserved among all strains examined (figure 2A). No cysteine residues
were affected by strain variation except at codon 15 within the signal sequence.
Discussion This analysis indicates that there are strong constraints on genetic variation in CMV gH. This conservation is consistent with an essential role for gH in the viral life cycle, as suggested by findings with the homologous herpes simplex gene product [11]. The two observed gH sequence groups are fewer than were noted in gB [4, 5]. The percentage ofvariant codons in gH is similar to that in the gp55 component of gB [5] but is less than that in gpl16 [4]. This sample ofstrains from a local patient population does not exclude the possibility that other major gH variants exist elsewhere. However, in a comparable study of gB variation, in which four major peptide sequence groups were defined among these local strains, sequence data from other parts of the world have so far revealed the existence of the same groups [4]. Of interest is that strains AD 169 and Towne, the sources of which are remote in space and time, easily segregated into the same groups as the local strains. Theoretically, there is also concern that propagation in cell culture selects for the two gH variant groups defined here. However, available data suggest that gH sequences amplified by PCR directly from clinical specimens are comparable to those amplified from their culture isolates [9]. The likelihood that PCR amplification misrepresents the sequences present in the viral DNA is reduced by the multiple independent reactions used to generate templates for each strain and the extensive and coherent overlap of the data obtained. The limited, group-specific diversity of both gB and gH
Downloaded from http://jid.oxfordjournals.org/ at Michigan State University on June 18, 2015
Forward (coding) strand
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Concise Communications
A II~UII
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i
I
1ID 1992; 166 (September)
•
I I
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I
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B gH Group 1:
g8
AD169
MRPGLPPYLTVFTVYLLSHLPSNRYGADAASEALDPHAFH
C074 C079 C325 C338 C352 C353
------------A--------------------------------F-----A--------------------------------S-----A--------------------------------F-----A----------P---------------------S-----A--------------------------------F-----A---------------------------
GTEISIP
HDWKGS
2 4 1 1 2
2 3
-ANV-V-ANV-V-ANV-V-ANV-V-ANV-V-
1 3
gH Group 2: Towne C076 C327 C336 C351 Codon
------S--I-LA-C-----L-S----E-I--P--KX--------S--I-LA-C-----L-P----E-V--P--KX--------S--I-LA-C-----L-P----E-V--P--KX--------S--I-LA-C-----L-P----E-V--P--KX--------S--I-LA-C-----L-P----E-V--P--KX--40
-G-TE-G-TE-G-TE-G-TE-G-TE-
176
must be reconciled with restriction analyses ofCMV isolates that reveal a virtually unlimited number of CMV strains. Although silent mutation at the nucleotide level accounts for some of this diversity, and there may be regions of greater mutability than gB and gH, variation in the genome as a whole is likely to be generated primarily by recombination and facilitated by the high frequency of infection of individuals by multiple strains ofCMV [10]. Lack ofconcordance of gH and gB grouping data on the clinical isolates screened for this study (figure 1B) suggests that considerable recombination has occurred in the 27-kb distance between the genes. The humoral immune response to CMV gH in naturally infected persons is variable and often weak, although it apparently rises to higher levels after secondary infections [12]. Whether a high titer of antibody to epitopes of this specific protein confers a protective effect remains to be established. Studies with murine and human monoclonal antibodies reveal conformational and nonconformational (linear) neutralizing epitopes in gH. Reactivity of human sera to fusion proteins expressing parts of CMV gH suggested that residues 34-43 constitute an important antibody binding site that is recognized in a strain-specific manner [13]: However, sequences presented here reveal only two discernable peptide configurations at this binding site. Immunologic reactivity to most strains can therefore be expected after exposure to the two types of gH; this simplifies the design of candidate subunit vaccines. A neutralizing murine monoclonal antibody recognizing a conformational epitope of gH reacted to cells transfected with full-length gH but not to those transfected with truncated versions lacking ~34 codons at the 3' end [14]. The conserved C-terminal part ofgH may participate in formation of conformational epitopes. The high degree ofconservation of nucleotide sequence in parts ofgB and gil makes them attractive genomic targets for
181
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1 2 1
365
diagnostic DNA amplification. With the data now available, it is possible to design primers of conserved sequence that amplify targets showing some group variability. This can be used to. distinguish strains and aid in the authentication of products amplified from clinical specimens [9].
Acknowledgments Marcia Kennedy, Taylor Dunn, and Gail Marousek provided technical assistance.
References I. Rasmussen L. Immune response to human cytomegalovirus infection. Curr Top Microbiol Immunol 1990; 154:221-54. 2. Kniess N. Mach M. Fay J. Britt WJ. Distribution of linear antigenic sites on glycoprotein gp55 of human cytomegalovirus. J Virol 1991;65: I38-46. 3. Meyer H. Masuho Y. Mach M. The gp116 complex of the gp58/116 complex of human cytomegalovirus represents the amino-terminal part of the precursor molecule and contains a neutralizing epitope. J Gen Virol 1990;71 :2443-50. 4. Chou S. Comparative analysis ofsequence variation in gp 116 and gp55 components of glycoprotein B of human cytomegalovirus. Virology 1992; 188:388-90. 5. Chou S. Dennison KM. Analysis of inters train variation in cytomegalovirus glycoprotein B sequences encoding neutralization-related epitopes. J Infect Dis 1991;163:1229-34. 6. Pachl C. Probert WS. Hermsen KM. et al. The human cytomegalovirus strain Towne glycoprotein H gene encodes glycoprotein p86. Virology 1989; 169:418-26. 7. Keay S. Merigan 'I'C, Rasmussen L. Identification of cell surface receptors for the 86-kilodalton glycoprotein of human cytomegalovirus. Proc Natl Acad Sci USA 1989;86:10100-3. 8. Aulitzky WE. Schulz TF. Tilig H. et al. Human monoclonal antibodies neutralizing cytomegalovirus (CMV) for prophylaxis of CMV disease: report of a phase I trial in bone marrow transplant recipients. J Infect Dis 1991;163:1344-7.
Downloaded from http://jid.oxfordjournals.org/ at Michigan State University on June 18, 2015
-A-----
Figure 2. Peptide sequence variation in CMV glycoprotein H. A, Map of gH codons showing positions of variation in 1 (shorter vertical lines) or > 1 (longer lines) of 12 strains sequenced. e, Potential glycosylation sites. B, Sequence alignments of clinical and laboratory CMV strains with strain AD 169. First 40 residues of gH are shown, followed by 2 smaller clusters of variation. AD 169 and Towne sequences are as published [6]. gB genotype of each strain was determined as previouslydescribed [5].
JID 1992; 166 (September)
Concise Communications
9. Chou S. Differentiation of cytomegalovirus strains by restriction analysis of DNA sequences amplified from clinical specimens. J Infect Dis 1990; 162:738-42. 10. Chou S. Reactivation and recombination of multiple cytomegalovirus strains from individual organ donors. J Infect Dis 1989; 160: 11-5. II. Forrester A, Farrell H. Wilkinson G, Kaye J, Poynter NO, Minson T. Construction and properties ofa mutant of herpes simplex virus type I with glycoprotein H coding sequences deleted. J Virol 1992;66:341-8.
607
12. Rasmussen L, Matkin C. Spaete R, Pachl C. Merigan TC. Antibody response to human cytomegalovirus glycoproteins gB and gH after natural infection in humans. J Infect Dis 1991;164:835-42. 13. Urban M, Britt W, Mach M. The dominant linear neutralizing antibody-binding site of glycoprotein gp86 of human cytomegalovirus is strain specific. J Virol 1992;66: 1303-11. 14. Spaete RR, Perot K. Scott PI, et al. CMV (Towne) glycoprotein H (gH) is complexed with GRP78 and GRP94. Landini MP. ed. Progress in cytomegalovirus research. Amsterdam: Elsevier. 1991:133-6.
Effect of Foscarnet Therapy on Human Immunodeficiency Virus p24 Antigen Levels in AIDS Patients with Cytomegalovirus Retinitis St. Luke'slRoosevelt Hospital Center. New York. New York; Departments ofMedicine. University ofSouthern California, Los Angeles. Huntington Memorial Hospital. Pasadena. and University of California. San Francisco. and Medical Service. San Francisco General Hospital. California; Department of Medicine. University of North Carolina. Chapel Hill; Division of Infectious Diseases, George Washington University Medical Center. Washington, DC; Department ofMedicine. University of Washington. Seattle; Department ofBiostatistics. Harvard School of Public Health, Boston. Massachusetts
Circulating human immunodeficiency virus (HIV) p24 antigen levels were measured in 22 AIDS patients who had detectable serum antigen at baseline after induction and maintenance therapy of foscamet for cytomegalovirus retinitis in phase Ijll multicenter trials, The HIV p24 antigen levels decreased from a baseline value of 199 ± 236 (mean ± SD) and 140 pgjmL (median) to 106 ± 218 and 28 pgjmL after 14 days of foscarnet induction therapy (60 mgjkg every 8 h). During chronic foscamet maintenance, there was a sustained decrease in mean HIV p24 antigen levelsbelowpre-foscarnet therapy baselineconcentrations for a median of 16 weeks after foscarnet induction. These results provide evidence for a sustained clinical antiretroviral effect of chronic foscamet maintenance therapy, consistent with a recent report that foscarnettreated AIDS patients live longer than ganciclovir-treated patients. The sodium salt of phosphono formic acid (foscarnet), an inhibitor of reverse transcriptase in a number of retroviruses, has been shown to inhibit reverse transcriptase of human immunodeficiency virus (HIV) in vitro. and additive or synergistic inhibition of HIV replication has been noted when zidovudine was combined with foscarnet [1-3]. In clinical trials, short-term foscarnet treatment of AIDS patients with cytomegalovirus (CMV) retinitis has been reported to de-
Received 30 December 1991; revised 13 April 1992. Informed consent was obtained from all patients. and human experimentation guidelines of the US Department of Health and Human Services were followed in the conduct of clinical research. Grant support: National Institutes of Health (AI-25902. AI-27663, AI25868, AI-30992. and RR-0046). Reprints or correspondence: Dr. Mohan M. Reddy. AIDS Clinical Trials Unit. St. Luke's/Roosevelt Hospital Center. 428 W. 59th St., New York. NY 10019. The Journal of Infectious Diseases 1992;166:607-10 © 1992 by The University of Chicago. All rights reserved. 0022-1899/92/6603-0023$01.00
crease HIV-1 isolation and circulating HIV p24 antigen levels [4-6]. Furthermore, combined zidovudine and foscarnet therapy results in decreased HIV p24 antigen levels in an additive manner, suggesting an enhanced antiretroviral activity when two reverse transcriptase inhibitors are combined [7]. The purpose of this study was to determine the effect of foscarnet on HIV p24 antigen levels during foscarnet maintenance therapy. We report the effect ofinduction and maintenance foscarnet therapy on circulating HIV p24 antigen levels in AIDS patients with CMV retinitis who were antigenemic at entry into two National Institute of Allergy and Infectious Diseases (NIAID)-sponsored multicenter trials.
Subjects and Methods The study population was 22 HIV p24 antigenemic AIDS patients with CMV retinitis enrolled in NIAID ACTG (AIDS Clinical Trials Group) protocol 093 (14 patients) and ACTG
Downloaded from http://jid.oxfordjournals.org/ at Michigan State University on June 18, 2015
Mohan M. Reddy, Michael H. Grieco, George F. McKinley, Dennis M. Causey, Charles M. van der Horst, David M. Parenti, Thomas M. Hooton, Roger B. Davis, and Mark A. Jacobson
CONCISE COMMUNICATIONS
Molecular Epidemiology of Envelope Glycoprotein H of Human Cytomegalovirus SUDweD Chou
Medical and Research Services. VA Medical Center. and Division of Infectious Diseases. Oregon Health Sciences University. Portland. Oregon
Envelope glycoproteins Band H (gB, gH) ofcytomegalovirus (CMV) are probably essential for viral entry into host cells and are important targets of human immune responses such as neutralizing antibody [I]. The major glycoprotein (gB) has been studied extensively and shown to contain epitopes in both its gp5 5 and gp I 16 components that are recognized by murine monoclonal and human neutralizing antibody [2, 3]. Sequence data indicate that interstrain variation in gB is strongly clustered at several distinct loci and that clinical strains adopt one of up to four group-specific peptide configurations at these loci [4, 5]. gH (gp86) was initially characterized by sequencing and reactivity with a neutralizing murine monoclonal antibody [6]. In its glycosylated form, it has a size of 86 kDa and appears to bind a 92.5-kDa cellular receptor [7]. Neutralizing human monoclonal antibodies have been produced to gH and have been entered into clinical trials [8]. The published AD 169 and Towne strain gH sequences show significant variation in the first 37 codons and scattered changes in the rest of the molecule [6]. The purpose of the present study was to determine the diversity of gH sequences among clinical strains as an indication of the probable cross-reactivity of immune responses directed to this molecule and to aid in the design of diagnostic primers and amplification targets.
Received 3 February 1992; revised 22 April 1992. Financial support: Department of Veterans Affairs research funds. Ten complete gH coding sequences have been submitted to Genbank, and the accession and strain numbers. respectively. are as follows: M94228. C074; M94229. C076; M94230. C079; M94231. C325; M94232. C327; M94233. C336; M94234. C338; M94235. C351; M94236. C352; M94237. C353. Reprints or correspondence: Dr. Sunwen Chou. Infectious Disease Section II IF. VA Medical Center. P.O. Box 1034. Portland OR 97207. The Journal of Infectious Diseases 1992;166:604-7 © 1992 by The University of Chicago. All rights reserved. 0022-1899/92/6603-0022$01.00
Methods Ten clinical CMV strains were selected for sequencing from a library of > 30 strains that had been screened for diversity by restriction analysis of various target sequences amplified bythe polymerase chain reaction (PCR) [9]. They were genetically distinct and obtained from epidemiologically unrelated transplant recipients. Clinical isolates appearing to contain multiple strains by restriction analysis were excluded. To avoid artifactual sequence change during passage, plaque purification was not attempted except for strains C325 and C327, which were plaquepurified directly from the urine specimen [10]. These plaque-purified strains were sequenced at passage 11; DNA from all other strains was extracted within 6 passagesof primary isolation. Complete gH coding sequences were determined by dideoxy chain termination reactions on PCR-amplified biotinylated templates after attachment to streptavidin-coated magnetic beads as previously described for gB [5]. A separate PCR was done to generate a template for each sequencing reaction by using a biotinylated primer based on reference sequences at or beyond either end of the gH coding sequence (figure 1) paired with a primer on the complementary strand, which was located in a 5' direction with respect to the intended sequencing primer. Overlapping coverage of the 2.2-kb coding sequence was achieved by using synthetic oligonucleotide primers of conserved sequence (AD 169 and Towne strains), spaced :::;300 basesapart and representing both DNA strands of the region sequenced (figure 1). Thus, numerous separate PCR amplifications, with a variety of primer pairs, were done on a given viral DNA extract to generate the complete gH coding sequence.
Results As was found with gB [5], sequencing of PCR-generated templates provided coherent overlapping data for each strain despite the use of unpurified amplification products generated separately for each sequencing reaction. Thus, the strains sequenced here each appeared to contain a single ge-
Downloaded from http://jid.oxfordjournals.org/ at Michigan State University on June 18, 2015
The complete envelope glycoprotein H (gH) coding sequences of 10 clinical strains of cytomegalovirus (CMV) were determined and compared with those of laboratory strains AD169 and Towne. Their translated peptide sequences segregated into two groups, exemplified by AD169 and Towne. Peptide variation was mostly group-specific and was clustered in the first 37 amino acids, including the signal sequence; in the rest of the molecule, there were scattered amino acid substitutions, usually in single residues. Compared with CMV envelope glycoprotein B, gH is more highly conserved among strains and may be expected to have limited immunologic diversity.
Concise Communications
1ID 1992; 166 (September)
FigUre 1. CMV glycoprotein H amplification and sequencing primers.
605
Reverse (noncoding) strand
Name
Sequence (5'-3')
Name
Sequence (5'-3')
gH175 gH190 gH203* gH299 gH571 gHII08 gHl153 gH1321 gH1471 gH1672 gH1873
CTCCTTCTCTCGGGTGTAAC AGCAGCCTCCGTAACAGCAC CCACCTGGATCACGCCGCTG GTCCTCTGGCGGAGCAGTT CACACTTTAACCAGACCTGTAT CGCCAGGCCGCACTCTT CGACTTCAACTACCTGGACC CTACGACAGATCGCCGA GTAGAAACGGGCCTCTG ACCATGCAACCAAGCAC TGCGAACTAACGCGCAA
gH172 gH495 gH622 gH803 gH1288 gH1688 gH2002 gH2184 gH2228* gH2373 gH2424*
TGGTGTTTTCACGCAGGAA AGGTATTGACAGATCAATGG GTAGGTGTTAAGTCTCTG AAGTTGTCGCGTTGATAGGG TGCTAATGCGTAGGCGAAGG GTGCTTGGTTGCATGGT ATGTACATGCACGACTCGGA CGATAGCGCGTAGACGGACAT CATGTCTTGAGCATGCGGTA ACCATCACACCGTGTGGCGT ACCCATAACAGTACCTCGTA
*Biotinylated primers used to generate sequencing templatesC)
notype, whereas attempted sequencing of isolates known to contain mixtures of strains has resulted in conflicting data from different sequencing reactions (data not shown). Of the 743 codons in gH, 157 (21 %) showed interstrain variation at the nucleotide level and 34 (4.6%) at the peptide level. At both levels, but especially at the peptide level, the coding sequences segregated into two groups. AD 169 and related strains formed group 1, with a coding sequence of 743 codons, while Towne and related strains formed group 2, with a coding sequence of 742 codons. Strains in the same group had ~8 amino acid differences; strains in opposite groups had '" 30. The numbering system used here is from group 1; the group 2 strains all had a deletion at codon 36. Of the 10 clinical strains sequenced, 6 were in group 1 and 4 were in group 2. Restriction enzyme (HhaI) digestion of a PCR-amplified target (primers gH203jgH 172) yielded one of two fragment patterns that identified the gH group of a given strain; by this analysis, 20 of 40 additional random clinical strains from our hospital were in group 1 and the rest were in group 2. Thus, gH groups 1 and 2 were about equally common. Figure 2A shows the distribution of variable codons that affected peptide encoding. Nearly all of the clustered variation was in the first 37 codons, 21 of which are the probable signal sequence. The remaining variable codons were widely scattered and usually varied in a group-specific manner, although a few isolated mutations specific to a single strain were noted (figure 2A). More than half of the amino acid variation involved some change in polarity of the residue involved (figure 2B). There were six potential glycosylation sites (Asn ...Ser or Asn ...Thr) that were completely conserved among all strains examined (figure 2A). No cysteine residues
were affected by strain variation except at codon 15 within the signal sequence.
Discussion This analysis indicates that there are strong constraints on genetic variation in CMV gH. This conservation is consistent with an essential role for gH in the viral life cycle, as suggested by findings with the homologous herpes simplex gene product [11]. The two observed gH sequence groups are fewer than were noted in gB [4, 5]. The percentage ofvariant codons in gH is similar to that in the gp55 component of gB [5] but is less than that in gpl16 [4]. This sample ofstrains from a local patient population does not exclude the possibility that other major gH variants exist elsewhere. However, in a comparable study of gB variation, in which four major peptide sequence groups were defined among these local strains, sequence data from other parts of the world have so far revealed the existence of the same groups [4]. Of interest is that strains AD 169 and Towne, the sources of which are remote in space and time, easily segregated into the same groups as the local strains. Theoretically, there is also concern that propagation in cell culture selects for the two gH variant groups defined here. However, available data suggest that gH sequences amplified by PCR directly from clinical specimens are comparable to those amplified from their culture isolates [9]. The likelihood that PCR amplification misrepresents the sequences present in the viral DNA is reduced by the multiple independent reactions used to generate templates for each strain and the extensive and coherent overlap of the data obtained. The limited, group-specific diversity of both gB and gH
Downloaded from http://jid.oxfordjournals.org/ at Michigan State University on June 18, 2015
Forward (coding) strand
606
Concise Communications
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1ID 1992; 166 (September)
•
I I
I
400
•
I
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I
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700
B gH Group 1:
g8
AD169
MRPGLPPYLTVFTVYLLSHLPSNRYGADAASEALDPHAFH
C074 C079 C325 C338 C352 C353
------------A--------------------------------F-----A--------------------------------S-----A--------------------------------F-----A----------P---------------------S-----A--------------------------------F-----A---------------------------
GTEISIP
HDWKGS
2 4 1 1 2
2 3
-ANV-V-ANV-V-ANV-V-ANV-V-ANV-V-
1 3
gH Group 2: Towne C076 C327 C336 C351 Codon
------S--I-LA-C-----L-S----E-I--P--KX--------S--I-LA-C-----L-P----E-V--P--KX--------S--I-LA-C-----L-P----E-V--P--KX--------S--I-LA-C-----L-P----E-V--P--KX--------S--I-LA-C-----L-P----E-V--P--KX--40
-G-TE-G-TE-G-TE-G-TE-G-TE-
176
must be reconciled with restriction analyses ofCMV isolates that reveal a virtually unlimited number of CMV strains. Although silent mutation at the nucleotide level accounts for some of this diversity, and there may be regions of greater mutability than gB and gH, variation in the genome as a whole is likely to be generated primarily by recombination and facilitated by the high frequency of infection of individuals by multiple strains ofCMV [10]. Lack ofconcordance of gH and gB grouping data on the clinical isolates screened for this study (figure 1B) suggests that considerable recombination has occurred in the 27-kb distance between the genes. The humoral immune response to CMV gH in naturally infected persons is variable and often weak, although it apparently rises to higher levels after secondary infections [12]. Whether a high titer of antibody to epitopes of this specific protein confers a protective effect remains to be established. Studies with murine and human monoclonal antibodies reveal conformational and nonconformational (linear) neutralizing epitopes in gH. Reactivity of human sera to fusion proteins expressing parts of CMV gH suggested that residues 34-43 constitute an important antibody binding site that is recognized in a strain-specific manner [13]: However, sequences presented here reveal only two discernable peptide configurations at this binding site. Immunologic reactivity to most strains can therefore be expected after exposure to the two types of gH; this simplifies the design of candidate subunit vaccines. A neutralizing murine monoclonal antibody recognizing a conformational epitope of gH reacted to cells transfected with full-length gH but not to those transfected with truncated versions lacking ~34 codons at the 3' end [14]. The conserved C-terminal part ofgH may participate in formation of conformational epitopes. The high degree ofconservation of nucleotide sequence in parts ofgB and gil makes them attractive genomic targets for
181
359
1 2 1
365
diagnostic DNA amplification. With the data now available, it is possible to design primers of conserved sequence that amplify targets showing some group variability. This can be used to. distinguish strains and aid in the authentication of products amplified from clinical specimens [9].
Acknowledgments Marcia Kennedy, Taylor Dunn, and Gail Marousek provided technical assistance.
References I. Rasmussen L. Immune response to human cytomegalovirus infection. Curr Top Microbiol Immunol 1990; 154:221-54. 2. Kniess N. Mach M. Fay J. Britt WJ. Distribution of linear antigenic sites on glycoprotein gp55 of human cytomegalovirus. J Virol 1991;65: I38-46. 3. Meyer H. Masuho Y. Mach M. The gp116 complex of the gp58/116 complex of human cytomegalovirus represents the amino-terminal part of the precursor molecule and contains a neutralizing epitope. J Gen Virol 1990;71 :2443-50. 4. Chou S. Comparative analysis ofsequence variation in gp 116 and gp55 components of glycoprotein B of human cytomegalovirus. Virology 1992; 188:388-90. 5. Chou S. Dennison KM. Analysis of inters train variation in cytomegalovirus glycoprotein B sequences encoding neutralization-related epitopes. J Infect Dis 1991;163:1229-34. 6. Pachl C. Probert WS. Hermsen KM. et al. The human cytomegalovirus strain Towne glycoprotein H gene encodes glycoprotein p86. Virology 1989; 169:418-26. 7. Keay S. Merigan 'I'C, Rasmussen L. Identification of cell surface receptors for the 86-kilodalton glycoprotein of human cytomegalovirus. Proc Natl Acad Sci USA 1989;86:10100-3. 8. Aulitzky WE. Schulz TF. Tilig H. et al. Human monoclonal antibodies neutralizing cytomegalovirus (CMV) for prophylaxis of CMV disease: report of a phase I trial in bone marrow transplant recipients. J Infect Dis 1991;163:1344-7.
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Figure 2. Peptide sequence variation in CMV glycoprotein H. A, Map of gH codons showing positions of variation in 1 (shorter vertical lines) or > 1 (longer lines) of 12 strains sequenced. e, Potential glycosylation sites. B, Sequence alignments of clinical and laboratory CMV strains with strain AD 169. First 40 residues of gH are shown, followed by 2 smaller clusters of variation. AD 169 and Towne sequences are as published [6]. gB genotype of each strain was determined as previouslydescribed [5].
JID 1992; 166 (September)
Concise Communications
9. Chou S. Differentiation of cytomegalovirus strains by restriction analysis of DNA sequences amplified from clinical specimens. J Infect Dis 1990; 162:738-42. 10. Chou S. Reactivation and recombination of multiple cytomegalovirus strains from individual organ donors. J Infect Dis 1989; 160: 11-5. II. Forrester A, Farrell H. Wilkinson G, Kaye J, Poynter NO, Minson T. Construction and properties ofa mutant of herpes simplex virus type I with glycoprotein H coding sequences deleted. J Virol 1992;66:341-8.
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12. Rasmussen L, Matkin C. Spaete R, Pachl C. Merigan TC. Antibody response to human cytomegalovirus glycoproteins gB and gH after natural infection in humans. J Infect Dis 1991;164:835-42. 13. Urban M, Britt W, Mach M. The dominant linear neutralizing antibody-binding site of glycoprotein gp86 of human cytomegalovirus is strain specific. J Virol 1992;66: 1303-11. 14. Spaete RR, Perot K. Scott PI, et al. CMV (Towne) glycoprotein H (gH) is complexed with GRP78 and GRP94. Landini MP. ed. Progress in cytomegalovirus research. Amsterdam: Elsevier. 1991:133-6.
Effect of Foscarnet Therapy on Human Immunodeficiency Virus p24 Antigen Levels in AIDS Patients with Cytomegalovirus Retinitis St. Luke'slRoosevelt Hospital Center. New York. New York; Departments ofMedicine. University ofSouthern California, Los Angeles. Huntington Memorial Hospital. Pasadena. and University of California. San Francisco. and Medical Service. San Francisco General Hospital. California; Department of Medicine. University of North Carolina. Chapel Hill; Division of Infectious Diseases, George Washington University Medical Center. Washington, DC; Department ofMedicine. University of Washington. Seattle; Department ofBiostatistics. Harvard School of Public Health, Boston. Massachusetts
Circulating human immunodeficiency virus (HIV) p24 antigen levels were measured in 22 AIDS patients who had detectable serum antigen at baseline after induction and maintenance therapy of foscamet for cytomegalovirus retinitis in phase Ijll multicenter trials, The HIV p24 antigen levels decreased from a baseline value of 199 ± 236 (mean ± SD) and 140 pgjmL (median) to 106 ± 218 and 28 pgjmL after 14 days of foscarnet induction therapy (60 mgjkg every 8 h). During chronic foscamet maintenance, there was a sustained decrease in mean HIV p24 antigen levelsbelowpre-foscarnet therapy baselineconcentrations for a median of 16 weeks after foscarnet induction. These results provide evidence for a sustained clinical antiretroviral effect of chronic foscamet maintenance therapy, consistent with a recent report that foscarnettreated AIDS patients live longer than ganciclovir-treated patients. The sodium salt of phosphono formic acid (foscarnet), an inhibitor of reverse transcriptase in a number of retroviruses, has been shown to inhibit reverse transcriptase of human immunodeficiency virus (HIV) in vitro. and additive or synergistic inhibition of HIV replication has been noted when zidovudine was combined with foscarnet [1-3]. In clinical trials, short-term foscarnet treatment of AIDS patients with cytomegalovirus (CMV) retinitis has been reported to de-
Received 30 December 1991; revised 13 April 1992. Informed consent was obtained from all patients. and human experimentation guidelines of the US Department of Health and Human Services were followed in the conduct of clinical research. Grant support: National Institutes of Health (AI-25902. AI-27663, AI25868, AI-30992. and RR-0046). Reprints or correspondence: Dr. Mohan M. Reddy. AIDS Clinical Trials Unit. St. Luke's/Roosevelt Hospital Center. 428 W. 59th St., New York. NY 10019. The Journal of Infectious Diseases 1992;166:607-10 © 1992 by The University of Chicago. All rights reserved. 0022-1899/92/6603-0023$01.00
crease HIV-1 isolation and circulating HIV p24 antigen levels [4-6]. Furthermore, combined zidovudine and foscarnet therapy results in decreased HIV p24 antigen levels in an additive manner, suggesting an enhanced antiretroviral activity when two reverse transcriptase inhibitors are combined [7]. The purpose of this study was to determine the effect of foscarnet on HIV p24 antigen levels during foscarnet maintenance therapy. We report the effect ofinduction and maintenance foscarnet therapy on circulating HIV p24 antigen levels in AIDS patients with CMV retinitis who were antigenemic at entry into two National Institute of Allergy and Infectious Diseases (NIAID)-sponsored multicenter trials.
Subjects and Methods The study population was 22 HIV p24 antigenemic AIDS patients with CMV retinitis enrolled in NIAID ACTG (AIDS Clinical Trials Group) protocol 093 (14 patients) and ACTG
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Mohan M. Reddy, Michael H. Grieco, George F. McKinley, Dennis M. Causey, Charles M. van der Horst, David M. Parenti, Thomas M. Hooton, Roger B. Davis, and Mark A. Jacobson
