1 Department of Pathology, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan
2 Department of Microbiology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
3 Feed Division, Livestock Industry Department Agricultural Production Bureau Ministry of Agriculture, Forestry and Fisheries, 1-2-1 Kasumigaseki, Chiyoda-ku, Tokyo 100-8950, Japan
4 Research Center for Biologicals, Kitasato Institute, 6-111 Arai, Kitamoto-shi, Saitama 364-0026, Japan
5 Laboratory of Prevention of Viral Diseases, Research Foundation for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan
Correspondence
Tomoki Yoshikawa
ytomoki{at}nih.go.jp
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ABSTRACT |
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INTRODUCTION |
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The molecular mechanism of protection against virus infection by Abs is known as neutralization, a process in which binding of Abs to virus can result in decreased viral infectivity. IgA neutralizes viral infectivity by forming virusIg complexes, which prevents either the attachment to or penetration into cultured epithelial cells by the virus or neutralizes the virus within the cultured cell (Outlaw & Dimmock, 1990, 1991
; Armstrong & Dimmock, 1992
). Secretory IgA has a higher in vitro neutralization efficiency than IgG due to its polymeric nature (Renegar et al., 1998
). An in vivo study showed that virusIgA and virusIgM immune complexes were detected in the faeces of newborn piglets infected with rotavirus at the time of virus clearance (Corthier & Vannier, 1983
). This finding supports the concept that Abs neutralize viral infectivity in vivo by forming virusIg complexes that result in clearance of infectious virus. The mechanism of virusIg complex formation in vivo and clearance of infectious virus from the respiratory tract is unknown.
The detection of influenza virus RNA in patients with respiratory tract infections using real-time RT-PCR has been developed recently (van Elden et al., 2001). Another recently developed method is the use of immunocapture RT-PCR, which has been used to detect hepatitis C virus (HCV) particles bound to different anti-HCV Ig isotypes: viral particles were captured using specific Abs and then amplified (Peng et al., 2001
). These highly sensitive methods are useful tools for measuring the total number of influenza virus particles and were used in this study to ascertain the number of viruses in influenza virusIgA complexes in nasal secretions of mice. The role of the virusIgA complexes in providing cross-protection against variant viral infections was investigated.
In the present study we investigated the kinetics of total influenza virus particle production and that of virus within virusIg complexes after infection with A/PR8 (H1N1), and analysed the kinetics of infectious virus (p.f.u.) production as an index of infectious virus levels in the nasal secretions of naive mice and mice immunized 4 weeks previously with the A/PR8, A/Yamagata (H1N1), A/Guizhou (H3N2) and B/Ibaraki strains of influenza virus. The total virus number and the number of viruses within the immune complexes, captured using anti-mouse Ig-coated plates, were estimated on the basis of a viral genome copy number determined by quantitative RT-PCR (Q-PCR) (Peng et al., 2001; van Elden et al., 2001
). The results showed that the total virus number, which was 103104-fold higher than the number of infectious virus particles, correlated with the number of p.f.u. identified in nasal secretions of naive and immunized mice. Analysis of the number of p.f.u. and the total virus number revealed earlier virus elimination from the nasal area in the immunized mice than in the naive mice. The rate of virus elimination increased with the level of antigenic relatedness between the immunizing and challenging viruses. The rate of virus elimination in immunized mice correlated with the level of A/PR8 virus-reactive Abs. Virus elimination was accompanied by the formation of virusIg complexes shortly after infection. These results suggested that the formation of virusIg complexes is involved in the rapid clearance of virus from the upper respiratory tract observed in immunized mice. Based on the results of this study, we have proposed a four-stage process of virus elimination to help explain the mechanism of virusIg complex formation in vivo and clearance of infectious virus from the respiratory tract.
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METHODS |
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Infection.
BALB/c mice (female, 6 weeks old; Japan SLC, Hamamatsu) were anaesthetized by intraperitoneal injection of amobarbital sodium (0·25 ml of a 1 µg ml1 solution) and immunized by intranasal application of 2 µl virus suspension. The nose-restricted volume (2 µl) of the virus suspension induced a transient infection localized to the upper respiratory tract and was not lethal (Yetter et al., 1980; Tamura et al., 1996
). The following doses of virus were used to induce an appropriate level of individual anti-influenza virus Ab response: 7·4x102 p.f.u. for A/PR8, 4·0x104 p.f.u. for A/Yamagata, 4·0x103 p.f.u. for A/Guizhou-X and 1·1x104 p.f.u. for B/Ibaraki. Four weeks after immunization, mice were infected with 7·4x105 p.f.u. A/PR8 virus.
Virus inactivation.
The A/PR8 virus was inactivated by UV irradiation and treated nine times with an autocross-link mode (120 000 µJ cm2 once) using a Stratalinker UV cross-linker 1800 (Stratagene). The UV treatment of live A/PR8 virus, capable of producing 7·4x105 p.f.u. per mouse in MadinDarby canine kidney (MDCK) cell culture, resulted in the complete loss of infectivity. This treatment resulted in only a slight reduction in total viral genome copy number from 7·4x107 to 1·3x107.
Nasal wash specimens.
Mice were anaesthetized and bled from the heart. The nasal wash was collected by washing the nasal cavity three times with the same 1 ml of PBS containing 0·1 % BSA (Tamura et al., 1992).
Ab titration.
IgA and IgG against haemagglutinin (HA) molecules purified from the A/PR8, A/Yamagata, A/Guizhou-X and B/Ibaraki viruses (Phelan et al., 1980) or those against whole virus particles of the A/PR8 virus were measured by ELISA as described previously (Tamura et al., 1992
). Briefly, ELISA was conducted sequentially from the solid phase (EIA plate; Costar) with a sequence of reagents consisting of the following: (i) purified HA molecules or rabbit anti-A/PR8; (ii) nasal wash; (iii) goat anti-mouse IgA (
-chain-specific; Southern Biotechnology Associates) or goat anti-mouse IgG (
-chain-specific; Jackson ImmunoResearch Laboratories) conjugated with biotin; (iv) streptavidin conjugated with alkaline phosphatase (Invitrogen Corporation) or streptavidin conjugated with
-galactosidase (Invitrogen Corporation); and (v) p-nitrophenylphosphate for alkaline phosphatase detection or 4-methylumbelliferyl-
-D-galactoside for
-galactosidase detection. The chromogen produced by alkaline phosphatase was measured for absorbance at 405 nm with a Labsystems Multiskan MS (Dainippon Pharmaceutical). The fluorescence produced by
-galactosidase was measured for excitation at 355 nm and emission at 460 nm with a Labsystems Fluoroskan II (Dainippon Pharmaceutical). Both A/PR8 HA-specific and A/PR8 virus-specific Ab levels were determined using a twofold serial dilution of purified HA-specific polyclonal IgA or HA-specific monoclonal IgG (starting at 160 ng ml1 each) as a standard. The Ab concentration of an unknown specimen was determined from the standard regression curve constructed for each assay. HA-specific Ab levels for viruses other than A/PR8 virus were determined using a twofold serial dilution of unknown nasal wash specimen, which was placed on the ELISA plate coated with the virus-specific HA molecules and expressed as the highest nasal wash dilution giving a positive reaction. The cut-off value was set as the mean+2 SD of a twofold serial dilution of pre-immune nasal wash specimens. The neutralization test was performed according to a standard method and the titre was represented by the serum dilution giving a 50 % reduction in number of p.f.u.
Plaque assay.
Serial 10-fold dilutions of the nasal wash in PBS with 0·1 % BSA were prepared and 0·2 ml aliquots were added to MDCK cells in a 6-well plate. After 1 h of adsorption, each well was overlaid with 2 ml 0·6 % agar medium (Tobita et al., 1975; Tobita, 1975
). After 2 days incubation in a CO2 incubator, the plaques were counted. The viral titre was expressed as p.f.u. ml1 and represented by the mean±SD of specimens collected from three mice of each group. Neutralization tests were carried out as follows. The A/PR8 virus suspension contained 50 p.f.u. in 0·1 ml 1 % BSA in PBS and was mixed with equal volumes of serial dilutions of antiserum and allowed to react at 37 °C for 1 h. Then 0·2 ml of the mixture was added to the MDCK culture in a 6-well plate, left to adsorb at 37 °C for 30 min, and washed with 5 ml PBS before adding the overlay for the standard plaque assay.
Electron microscopy (EM) particle counts of influenza virus.
Virus particles were counted by the loop-drop method (Watson et al., 1963; Shiraki et al., 1991
). Briefly, the virus solution was mixed with a standard latex particle solution (Stadex, 100 nm diameter; JSR Corporation). The mixture was placed on a grid and stained with 1 % neutral phosphotungstate. The number of virus and latex particles was counted using an electron microscope and the virus particle count was determined by comparison with the latex particle count in the standard solution.
Extraction of viral RNA from virusIg complexes in the nasal wash.
Total viral RNA in the nasal wash (250 µl) was extracted with Trizol (Invitrogen Corporation) according to the manufacturer's instructions. The virusIg complexes between virus and antiviral IgA, IgG or Ig(A/G/M) Abs in the nasal wash were separated as reported by Peng et al. (2001). Briefly, goat anti-mouse IgA (
-chain-specific; Southern Biotechnology Associates), goat anti-mouse IgG (
-chain-specific; Southern Biotechnology Associates) or goat anti-mouse Ig(A/G/M) (Zymed) was coupled to EIA plates (Costar). After washing with 0·05 % Tween 20 in PBS (PBS-Tween) and blocking with 1 % BSA in PBS, the plates were incubated with 50 µl nasal wash to capture the immune complexes present in the wash. After washing with PBS-Tween, viral RNA was extracted by incubating with 20 µl TE buffer (pH 8·0) and 40 µl Trizol according to the manufacturer's instructions.
Quantification of viral matrix protein gene copies.
Extracted viral RNA was reverse-transcribed into cDNA using a Sensiscript RT kit (Qiagen) containing a primer to the nucleotide sequence of the matrix protein (M1) gene (van Elden et al., 2001). The cDNA was amplified with 20 µl of a PCR mixture containing 2 µl cDNA, 2 µl LightCycler DNA Master Hybridization Probes (Roche Diagnostics), 5 mM MgCl2, 900 nM M1 gene-specific primers (INFA-1 and INFA-2) and 200 nM M1 gene-specific TaqMan probe (INFA probe) (van Elden et al., 2001
). Amplification and detection of cDNA were performed using a real-time Q-PCR system (LightCycler; Roche Diagnostics) under the following conditions: 10 min at 95 °C to activate the DNA polymerase, followed by 45 cycles of 0 s at 95 °C, 5 s at 55 °C and 10 s at 72 °C. Standard RNA was synthesized from plasmid DNA encoding the A/PR8 M1 gene by T7 RNA polymerase (Ribo Max Large Scale RNA Production Systems T7; Promega) and used as the standard to calculate the copy number after spectrophotometric determination of RNA concentration.
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RESULTS |
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Specificity of the detection system for immune complexes
The specificity of the detection system for immune complexes was examined. First, 10-fold serial dilutions of immune complexes (diluted from 1 : 100 to 1 : 108) formed between anti-A/PR8 HA mAb (160 ng ml1) and the A/PR8 virus (7·4x105 p.f.u. per mouse in MDCK cell culture) were incubated with goat anti-mouse IgG-coated ELISA plates. The viral genome copy number in the total RNA, extracted from the virusIg complexes, was then measured by Q-PCR. As a control, serial dilutions of the complexes were incubated with BSA-coated plates. As another control, the virus number was measured for the total RNA extracted directly from the diluted viruses, without prior incubation with goat anti-mouse IgG-coated plates. The number of viral genome copies recovered from the immune complexes decreased in a linear fashion with dilutions of the immune complexes, both represented on a log scale, when the immune complexes contained more than 103 viral genome copies (data not shown). Thus, virus could be recovered completely from the immune complexes bound to goat anti-mouse IgG-coated plates. In addition, non-specific binding of immune complexes to BSA-coated plates, which corresponded to approximately 0·10·01 % of the viral genome copy number, was observed at dilutions of the immune complexes that contained more than 105 viral genome copies (data not shown). This result showed that the viral genomes in the virusIg complexes could be detected specifically by Q-PCR.
Next, 10-fold serial dilutions of anti-A/PR8 HA mAb (10 µg ml1) (diluted up to 1 : 108) were incubated with the A/PR8 virus (1·1x105 copies ml1, 2·2x103 p.f.u. ml1) to form immune complexes and incubated with goat anti-mouse IgG-coated ELISA plates. The viral genome copy number in the virusIg complexes was measured by Q-PCR and shown to increase in the presence of 102 ng mAb ml1, reaching a plateau of 104105 copies in the range of 1 ng to 10 µg mAb ml1 with a low peak at 100 ng mAb ml1 (data not shown). Thus, a high viral copy number was involved in immune complex formation for a wide range of antibody concentrations. This made it difficult to detect slight differences in virus number within the immune complexes, depending on the level of antibody. On the other hand, an infectious virus response of 103 p.f.u. ml1 was produced in MDCK cell culture after incubation of the virus with 0·0011 ng mAb ml1. This response decreased when 100 ng mAb ml1 was used and disappeared at 10 µg mAb ml1 (data not shown). In addition, the supernatant collected from the plate after incubation of the mixture of the virus and the mAb (100 ng ml1) with the anti-mouse IgG-coated ELISA plate decreased the ability to produce infectious virus (1/10 the number of p.f.u. of the mixture). These results suggested that virusIg complex formation is involved in the reduction in the amount of infectious virus produced.
Number of p.f.u., total viral genome copy number and virusIg complex formation after infection with A/PR8 virus in naive mice and mice previously immunized with A/PR8 virus
Following A/PR8 virus infection of naive mice and mice immunized 4 weeks previously with the A/PR8 virus, the kinetics of infectious virus and total viral genome copy numbers were examined in nasal secretions. In the naive mice, the number of p.f.u. after infection decreased rapidly from 0 to 3 h, gradually increased from 3 to 12 h, peaked on day 3 at 104 p.f.u. (ml nasal wash)1 and declined thereafter (Fig. 1a and b). Similarly, the total virus number decreased rapidly within 3 h of infection, then increased to a peak on day 3 with 108 copies (ml nasal wash)1 and declined thereafter (Fig. 1c and d
). In the immunized mice, the number of infectious virus particles decreased rapidly from 0 to 3 h, increased to a peak at 12 h at 102 p.f.u. (ml nasal wash)1 and then disappeared within 24 h (Fig. 1a and b
). The total virus number decreased rapidly from 0 to 3 h, increased to a peak of 106 copies at 12 h, then disappeared on day 5 (Fig. 1c and d
). From 3 to 12 h after infection, the number of infectious virus particles in the immunized mice [10 p.f.u. (ml nasal wash)1] was lower than observed in the naive mice [102 p.f.u. (ml nasal wash)1], although the total virus number in immunized and naive mice was almost the same. In summary, the total virus number was 103104-fold higher than the number of infectious particles in both the naive and immunized mice, and total virus persisted in nasal secretions 46 days longer than the infectious virus. In the immunized mice, the challenge virus was eliminated within 24 h based on the number of infectious particles and within 5 days based on the total virus number. In naive mice, challenge virus elimination occurred 8 days later based on the number of infectious particles and 10 days later based on the total virus number (Fig. 1a and d
, and data not shown).
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The early elimination of challenge virus observed in immunized mice compared with naive mice (Fig. 1a and b) could be explained by the presence of high levels of nasal anti-A/PR8 virus or HA-specific Abs and neutralization Abs (Table 1
). To confirm this, the viral genome copy number within virusIg complexes after infection with A/PR8 virus was examined in the nasal secretions of immunized mice (Fig. 1e
). The number of viral genome copies bound to the specific IgA, IgG or Ig(A, G and M) Abs was detected immediately after infection. Viral genome copy number decreased within 3 h, increased to a peak at 12 h and then declined slowly up to 24 h. The virus number within virusIgA complexes (Fig. 1e
) was similar to that within virusIgG or virusIg(A/G/M) complexes (data not shown). We also observed that the change in the number of viruses within virusIgA complexes was slightly lower than the total virus number. These results suggested that homologous challenge virus was captured by specific Abs immediately after infection in A/PR8 virus-immunized mice, resulting in the formation of virusIg complexes (Fig. 1a and d
).
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DISCUSSION |
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Based on the present results, the process of virus clearance from the upper respiratory tract of mice after infection can be divided into four stages. In stage 1, which extends from 0 to 3 h after infection, both infectious and UV-inactivated viruses were eliminated rapidly in both the naive and the immunized mice (Fig. 1c and e). Total virus number decreased to 104105 copies. This rapid decrease is likely to be the result of non-specific elimination of virus via physiological cleaning systems in the nose, such as the ciliary movement of the epithelial cells of the nasal mucosa (Wilson et al., 1987
; Cone, 1999
). At the same time, the number of infectious virus particles in immunized mice decreased from 7·4x105 to 10 p.f.u. (ml nasal wash)1, which was lower than that observed in naive mice (Fig. 1a
). The lower number of infectious virus particles in the immunized mice 3 h after infection was most likely the result of neutralization of infectious virus in the presence of virus-reactive Abs. The importance of neutralization in reducing infectious virus levels was reflected in the formation of immune complexes between challenge virus and existing Abs (Fig. 1e
). Thus, it can be concluded that immune complexes are involved in the clearance of infectious virus in immunized mice. The discrepancy between the total virus number 012 h after challenge with infectious virus (106 copies) and that observed after administration of UV-inactivated virus (103 copies) (Fig. 1c
) suggested that susceptible host cells were infected by a small number of viruses (Lamb & Krug, 2001
).
In stage 2, which extends from 3 to 12 h after infection, the total virus number, infectious virus number and the virus number within the immune complexes increased after virus infection, although the total virus number decreased to its lowest level at 12 h (about 103 copies) after administration of UV-inactivated virus (Fig. 1c). These results may be explained by an increase in the number of progeny virus released from infected epithelial cells and an increase in the number of immune complexes formed between progeny virus and the existing Abs. Progeny virus may be released from infected epithelial cells 3·56 h after infection and continue to be released over several hours until the infected epithelial cells die (Reinacher & Weiss, 1975
; Lamb & Krug, 2001
). The progeny virus must be captured by existing IgA Abs in nasal secretions, which are secreted across the epithelial cells of the nasal mucosa, and IgG Abs, released from the serum by diffusion (Mestecky & McGhee, 1987
; Murphy, 1994
). Infectious virus was produced when a high titre of virus (7·4x105; Fig. 1
), but not a low titre of virus (7·4x102; data not shown), was used as the challenge infection dose in immunized mice. Thus, under the infection conditions with the high-titre virus, not all virus was neutralized by existing Abs, leading to infection of host cells. The degree of infectious virus development was lower in immunized mice than in naive mice, which had no pre-existing Abs (Fig. 1a
). However, the difference in the total virus number between immunized and naive mice was not as marked as the difference in the number of infectious virus particles. This may be the result of free viral genome copies and viral genome copies within the immune complexes persisting in nasal secretions for the first 12 h after infection so that the contribution of existing Abs in reducing total virus numbers is not detected.
In stage 3, which extends from 12 h to 3 days after infection, total virus, infectious virus and virus within immune complexes all decreased slowly in number. The degree of reduction of total and infectious virus number correlated with the level of anti-A/PR8 virus-reactive Abs observed in A/PR8, A/Yamagata and A/Guizhou-X virus-immunized mice, in decreasing order (Table 1; Fig. 1
). The immune mechanisms providing protection against influenza virus infection are redundant at this stage of infection (Couch & Kasel, 1983
; Murphy & Clements, 1989
; McMichael, 1994
). It has been reported that influenza virus-specific CTL responses are involved in the clearance of viruses from the lung and nose and that the CTL responses appear 3 days after a secondary infection (Yap & Ada, 1978
; Flynn et al., 1998
; Wiley et al., 2001
). This observation that CTL activity was detected only after the third day of secondary infection in the different subtype virus-immunized mice suggested that pre-existing Abs in the A/PR8 and the A/Yamagata virus-immunized mice were involved in the elimination of challenge viruses by forming virusIg complexes. This was shown to occur within 24 h and 3 days, respectively, in A/PR8 and the A/Yamagata virus-immunized mice based on estimations of the number of infectious virus particles following infection (Fig. 1
; Table 1
).
In stage 4, which extends from day 3 onwards after infection, the amount of infectious virus decreased slowly and disappeared within 4 days after A/PR8 virus infection of A/Guizhou-X virus-immunized mice. CTL memory cells are involved in the clearance of virus-infected cells in mice immunized with different subtype viruses (Wilson et al., 1987; Murphy & Clements, 1989
; McMichael, 1994
; Wiley et al., 2001
). Thus, in mice immunized with different subtype viruses, secondary CTL responses accelerated by the challenge infection may be involved in virus clearance from day 3 after infection. Investigations are being carried out to define further the events in this stage.
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Received 11 December 2003;
accepted 26 April 2004.
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