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Complement C4 Deficiency and HLA. Homozygosity in Patients with Frequent Intraoral Herpes. Simplex Virus Type 1 Infections. Mikko Seppänen,1 Marja-Liisa ...
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Complement C4 Deficiency and HLA Homozygosity in Patients with Frequent Intraoral Herpes Simplex Virus Type 1 Infections Mikko Seppa¨nen,1 Marja-Liisa Lokki,3 Tuomo Timonen,4 Maija Lappalainen,2 Hanna Jarva,5 Asko Ja¨rvinen,1 Seppo Sarna,6 Ville Valtonen,1 and Seppo Meri5 1

Department of Medicine, Division of Infectious Diseases, and 2Laboratory Diagnostics, Department of Virology, Helsinki University Central Hospital; 3 Department of Tissue Typing, Finnish Red Cross Blood Transfusion Service; 4 Department of Pathology, Haartman Institute; and 5Department of Bacteriology and Immunology and 6Department of Public Health, University of Helsinki, Helsinki

Three consecutive patients with no apparent immunodeficiency who had frequent intraoral herpes simplex type 1 recurrences, a rare complication of herpes simplex virus infection, were found to have a total deficiency of either the A or B isotype of the complement component C4 and to be homozygous for the studied HLA antigens. A combination of HLA homozygosity, which may lead to impaired T cell recognition of viral peptides, and deficiency in the classical complement pathway, which can compromise virus neutralization, may predispose to severe and frequent herpes simplex virus infections. Morbidity from herpes simplex virus type 1 (HSV-1) infection mainly stems from the ability of HSV-1 to periodically reactivate. Recurrent herpes labialis occurs in 15%–40% of seropositive persons [1]. Immunocompetent persons rarely have intraoral lesions. Of those who do, 52% experience recurrent episodes, commonly misdiagnosed as aphthous stomatitis or Received 16 March 2001; revised 31 May 2001; electronically published 26 September 2001. Written informed consent was obtained from all the patients. The study protocol was approved by the Ethics Committee of the Department of Medicine, Hospital District of Helsinki and Uusimaa and the human experimentation guidelines of the author’s institutions were followed in the conduct of clinical research. Financial support: University of Helsinki, Helsinki University Central Hospital Funds, Finnish Red Cross Blood Transfusion Service, Sigrid Juse´lius Foundation, Academy of Finland. Reprints or correspondence: Dr. Mikko Seppa¨nen, Division of Infectious Diseases, Dept. of Medicine, Helsinki University Central Hospital, Aurora Hospital, Ward 4, 2d floor, P.O. Box 348, FIN-00029 HUS, Finland ([email protected]). Clinical Infectious Diseases 2001; 33:1604–7  2001 by the Infectious Diseases Society of America. All rights reserved. 1058-4838/2001/3309-0026$03.00

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erythema multiforme [2–4]. Lesions occur predominantly on the hard palate and gingiva and can effectively be treated with long-term antiviral prophylaxis. The efficiency of the early immune response to clear HSV1 varies greatly [5]. Differences in IFN-g and specific antibody production and abnormalities in macrophage or NK cell function are thought to account for the differences in recurrence rates and invasiveness. HSV-1 has a complement evasion molecule that almost completely blocks the alternative complement pathway and terminal complex activation locally [6]. Virus neutralization by the host probably depends on antibody-induced activation of the classical pathway. Still, the role of complement deficiencies in HSV infections has received little attention [7]. Within 1 year, 3 unrelated patients from nonconsanguineous marriages were referred to us because of exceptionally active intraoral herpes with no apparent immunodeficiency. We studied complement functions and complement factor C4 and HLA genes in this small Caucasian patient group. The results point out immunologic features that may predispose to active intraoral HSV-1 infection. Methods. These 3 female patients had intraoral HSV-1 infection with 110 recurrences yearly, with recurrent herpetic blisters predominantly in the hard palate and gingiva. The recurrence rate had remained constant or increased over time (table 1). Blood was drawn into tubes containing heparin, EDTA (10 mM), or no anticoagulant. The samples were taken while the patients were asymptomatic. Serum antibodies to HSV-1 and HSV-2 were measured by commercial EIAs (HSV-1 ELISA IgG and HSV-2 ELISA IgG; MRL Diagnostics). The samples for HSV PCR, antigen detection, and isolation were taken from the bases of newly punctured intraoral vesicles. HSV isolation and type-specific PCR analyses were done [8–10]. The samples for HSV antigen detection were prepared from the swabs by cytocentrifugation [11]. Slides were stained with polyclonal HSV anti-rabbit IgG followed by fluorescein isothiocyanate–labeled anti-rabbit IgG (Jackson ImmunoResearch Laboratories). Paraffin-embedded tissue samples were stained similarly. The levels of IgG, IgA, and IgM and the concentrations of IgG subclasses were quantified by nephelometry with polyclonal antibodies from Behringwerke. DNA samples were genotyped for HLA-A, -B, -C, and -DR by means of the commercial kits of Pel-Freez (PF-ABC-SSP), One Lambda (SSP ABDR), or INNO-Lipa (LiPA HLA-DRB1) with low to intermediate allele resolutions. Complement C4

Table 1. Features of herpes simplex virus (HSV) infections in 3 women with recurrent intraoral HSV infections and results of virological and immunologic examinations. Feature

Patient 1

Patient 2

Patient 3

Clinical data Age, years

42

Age at first clinical herpes, years

20

9

5

Age when worsened, years

38

21

13

Yearly recurrences Site of lesions

26

11

20

Gingiva, hard palate, labia, retropharynx

Gingiva, hard palate

47

12 Gingiva, hard palate, labia

HSV analyses HSV antibodies, EIA units HSV-1 IgG

179

193

144

HSV-2 IgG

!20

!20

!20

HSV verification HSV-1 PCR

Positive





Histology, HSV-specific immunostaining



Positive



HSV-1 isolation, antigen detection





Positive

Complement analyses Serum levels, g/La C3

0.97

1.28

1.04

C4

0.14

0.09

0.08

Hemolytic activity Classical pathway, IU/mLb

!20

40

!20

95

1126

95

Alternative pathway, %c NOTE. a b c

Boldface type indicates abnormal results.

Reference values, 0.5–1.5 g/L for C3 and 0.15–0.5 g/L for C4. Reference values, 50–130 IU/mL. Percent of activity of control.

allotypes were determined electrophoretically [12]. The absence of an isotype was confirmed by isotype-specific PCR amplification of the C4A and C4B genes [13]. Serum C3 and C4 levels were quantified by nephelometry with use of polyclonal antibodies and reference samples from Behringwerke. The classical and alternative pathway hemolytic complement activities were determined by standardized hemolysis-in-gel assays from Binding Site. Activities of peripheral blood mononuclear cells (PBMC) were measured by incorporation of [3H] thymidine after stimulation of PBMC with phytohemagglutinin, concanavalin A, and pokeweed mitogen and were found to be normal (data not shown) [14, 15]. Results. All patients had 110 annual HSV-1 recurrences (table 1) that lasted 5–10 days but were completely suppressed with long-term oral valacyclovir therapy. The symptomatic relief started in !24 h. The patients had multiple small (1–3 mm diameter) vesicles at sites typical for intraoral HSV infection (table 1) but atypical for intraoral erythema multiforme or aphthous stomatitis. Patients 1 and 3 frequently manifested concurrent typical herpes labialis (table 1). HSV-1 infection

had become more frequent during pregnancy in patient 1 and at puberty in patient 3. Both also had signs of meningeal irritation with nuchal rigidity and myalgia, photophobia, and incapacitating headache at the same time as herpetic lesions. No increased protein level or pleocytosis in CSF were detected, and the PCR test for HSV in CSF was negative for patient 1. Patients 1 and 3 had frequent upper respiratory tract infections and patient 1 had recurrent sinusitis. The patients had no cytopenias or antibodies to HIV or hepatitis B or hepatitis C viruses. The results of virological and immunologic examinations are shown in tables 1 and 2. All patients were seropositive for HSV-1. Histological diagnosis for patient 2 was based on typical HSV blister morphology and confirmed by HSV-specific immunostaining. All had ongoing intraoral HSV-1 infections (table 1) and reduced classical complement pathway hemolytic activities and levels of serum C4 (table 1). C4 phenotyping showed a complete lack of the C4A (1 patient) or the C4B (2 patients) protein. Patients were homozygous for the HLA class I and II alleles (table 2). Patient 2, who was homozygous for A1,B8,DR3, had a low serum IgA level (0.47 g/L). BRIEF REPORTS • CID 2001:33 (1 November) • 1605

Table 2. Results of major histocompatibility complex typing for 3 women with recurrent intraoral herpes simplex virus (HSV) infections. Class II

Class I Patient

A*

B*

Cw*

DRB1*

1

03,03

35,35

04,04

2

01,01

08,08

07,07

3

02,02

15(62),15(62)

03(w10),03(w10)

NOTE.

Class III C4A

C4B

01,01

2,2

Q0,Q0

03,03

Q0,Q0

1,1

08,08

3,3

Q0,Q0

Q0, “null” allele.

Discussion. In our selected group of 3 patients, all had exceptionally active intraoral HSV-1 infections, were homozygous for their major histocompatibility complex (MHC) haplotypes, and had a total lack of either the C4A or C4B protein of the classical complement pathway (table 2) [16]. The patients thus have a combination of subtle disturbances in their MHC proteins that may compromise immune defense against exacerbations of HSV-1 in the oropharynx. Class I–restricted CD8⫹ T cells are thought to be responsible for the clearing of HSV. The variable rates of shedding and recurrence and the variation in the effectiveness of the early immune response is the net result of a complex interplay between the immune evasion molecules of HSV and the immune response of the patient [5]. It is likely that in addition to T cells, rapidly acting arms of the immune response contribute to this early stage of defense. NK, mononuclear, and CD4⫹ T cells, immunoglobulins, complement factors, and predominantly Th1-type cytokines are found in early HSV lesions. The first lymphocytes to migrate into HSV lesions are CD4⫹ T cells and NK cells, being responsible for the locally increased cytokine production. Products of the MHC class I HLA-A and -B loci present peptides to cytotoxic T and NK T cells and those of the class II loci to CD4⫹ T cells. HSV-1 can interfere with antigen presentation by class I and II MHC molecules and affect CD8⫹ and CD4⫹ T cell recognition [5]. As suggested in other viral infections, the expression of viral peptides to cytotoxic CD8⫹ T cells early during a recurrence may be hampered by HLA homozygosity [17], possibly through reduced variability in the patients’ HLA antigen repertoire, for example, on mucosal epithelial cells. Eventually, effects of cytokine production will overcome the inhibition of HLA class I expression by HSV, enabling the CD8⫹ T cells to clear the virus-infected cells [5]. MHC homozygosity has been linked to an accelerated disease progression in viral infections [17]. This is, to our knowledge, the first study to report an association between HLA homozygosity and symptomatic HSV recurrences. Both the B8,DR3- and B35,Cw4-containing MHC haplotypes have been implicated in recrudescent HSV disease and suggested to lead to an aggressive disease caused by enveloped

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viruses [18, 19]. The haplotypes that include the antigens A3,B35, A1,B8, and B15 occur in homozygote form in the Finnish population at frequencies 0.8%, 0.3%, and 2%, respectively [20]. Common to the patients’ haplotypes are C4 deletions. C4 protein has 2 isotypes, C4A and C4B, the genes of which are encoded in the MHC class III region. After disruption of their internal thiolester bonds, the C4A and C4B proteins bind covalently to amino or hydroxyl groups on target molecules and act as subunits in the classical pathway C3 convertase, C4b2a. C4 has a complex structural polymorphism with 140 protein allotypes recognized. C4 deficiency is caused by segmental deletions, duplications, gene conversions, or point mutations resulting in “null” (Q0) alleles. These occur with high frequency. About 35% of persons of all races lack 1 of the 4 C4A or C4B alleles [21]. Total C4A and C4B deficiencies, that is, 2 null alleles in the respective C4A or C4B gene loci, occur in 3% and 8% of the Finnish population, respectively [22]. The prevalence of intraoral herpes is unknown. We conservatively estimate it to be !0.5%. In the Finnish population, the prevalences of HLA homozygosity for the 9 most common haplotypes is 3% and for total C4A or C4B deficiency 11%. The prevalence of all these combined would thus be 1.65 per 100,000 persons, but HLA haplotypes and C4 deficiencies are not independent while linked. The probability that 3 consecutive patients with intraoral herpes would have these combined is minimal. As an example, among 10,000 consecutive hospitalized patients, the probability of finding 3 concordant patients, according to Poisson’s distribution, would be 0.00064. Specific antibodies and complement act in concert to directly neutralize and opsonize HSV and to augment antibody-dependent cellular cytotoxicity. To evade these rapidly acting arms of immunity, HSV-1 encodes specific proteins. First, IgFc-binding proteins inhibit IgG activity and enable the virus and infected cells to escape antibody attack [6]. Secondly, the HSV1 transmembrane glycoprotein gC-1 protects the virion and the HSV-infected cells against complement neutralization, thereby facilitating local spread [6]. Although the virus seems to be capable of coping with the alternative complement pathway, host resistance appears to depend on the classical pathway.

Deficiencies in it, or in antibodies activating it, could have a profound influence on the local spread and rates of recurrence of HSV-1. In conclusion, our study suggests that a deficiency of either of the 2 C4 proteins could be important in defining susceptibility to chronically active HSV-1 disease. In addition, HLA homozygosity also seems to be a susceptibility factor. Susceptibility to an exceptionally severe viral infection may thus be dictated by a combination of subtle defects in multiple arms of the immune system.

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