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1 Servizio di Virologia, Fondazione IRCCS Policlinico San Matteo, 27100 Pavia, Italy
2 Dipartimento di Medicina, Chirurgia e Odontoiatria, Università degli Studi di Milano, 20142 Milano, Italy
3 Laboratori Sperimentali di Ricerca, Fondazione IRCCS Policlinico San Matteo, 27100 Pavia, Italy
Correspondence
Giuseppe Gerna
g.gerna{at}smatteo.pv.it
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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128-fold higher neutralizing potency against a human cytomegalovirus (HCMV) clinical isolate propagated and tested in endothelial (or epithelial) cells than against the same virus infecting human fibroblasts. In a group of 18 primary infections, the reverse geometric mean titre was in the range of 10–15 in human fibroblasts within the first 3 months after the onset of infection, whereas the endothelial cell infection-neutralizing activity was already present within the first 10 days, reaching median levels of 122, 320 and 545 at respectively 30, 60 and 90 days after onset, then declining slowly. This difference was also confirmed in the majority of reactivated and remote HCMV infections, as well as in a hyperimmune globulin preparation. The antibody response to HCMV pUL131A, pUL130 and pUL128 locus products, which are required for endothelial/epithelial cell infection, provided a potential molecular basis for such a differential neutralizing activity. In addition, monoclonal/monospecific antibodies raised against the pUL131A, pUL130 and pUL128 proteins were found to display an inhibitory activity on HCMV plaque formation and HCMV leukocyte transfer from HCMV-infected cells. Hence, conventional determination of the neutralizing activity of human sera in fibroblasts is misleading. Antibodies to pUL131A, pUL130 and pUL128 appear to display a major HCMV-neutralizing and dissemination-inhibiting activity. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Revello & Gerna, 2002). On the other hand, although at a much lower rate, vertical transmission may also occur in seropositive pregnant women in the presence of neutralizing antibody (Fowler et al., 2003). Similarly, in immunocompromised patients, clinical syndromes occurring in seronegative individuals are more severe than those observed in seropositive subjects (Grossi et al., 1995; Gerna et al., 1998). However, reactivated infections occur in seropositive patients, even in the presence of high levels of neutralizing antibody (Muñoz et al., 2001). In addition, in these patients, HCMV infections are controlled only after reconstitution of cellular immunity (Quinnan et al., 1982; Reusser et al., 1991; Sester et al., 2005; Gerna et al., 2006a; Lilleri et al., 2006).
HCMV was recovered for the first time in 1956 in human fibroblasts and, since then, neutralizing antibodies have been evaluated in this cell system (Smith, 1956). Determination of neutralizing antibody has been proposed as a parameter for differentiating primary from reactivated HCMV infections, in that neutralizing antibodies appear on average at 13 weeks (range, 10–17 weeks) after onset of primary infection, whereas they are consistently detected early in reactivated infections (Eggers et al., 1998, 2000). Similarly, the kinetics of the glycoprotein B (gB) and glycoprotein H (gH) antibody responses were reported to resemble those of neutralizing antibody (Schoppel et al., 1998; Eggers et al., 2001).
More recently, the UL131A–128 locus of the HCMV genome has been shown to be indispensable for the infection of endothelial cells (Hahn et al., 2004), as well as epithelial cells (Wang & Shenk, 2005a). More extensively, the virion glycoprotein complex gH–gL–pUL131A–pUL130–pUL128 has been shown to be required for infection of both endothelial and epithelial cells, whilst the glycoprotein complex gH–gL–gO is required for infection of human fibroblasts (Wang & Shenk, 2005a). On this basis, it was decided to test human sera from different types of HCMV infection for neutralizing antibody in both endothelial and epithelial cells, in comparison with human fibroblasts, to verify whether the neutralizing activity is different in the two cell systems.
In this study, a striking difference was observed between endothelial and fibroblast cells in revealing the neutralizing activity of human sera from both subjects with primary and those with reactivated HCMV infections. Levels of neutralizing antibody were far higher when measured in endothelial (or epithelial) cells. In addition, these antibodies were shown to inhibit HCMV plaque formation and virus transfer from HCMV-infected cells to leukocytes, thus displaying a wide range of protective activity of the neutralizing-antibody response during natural infection. These results indicate the need for a revision of the approach adopted thus far for HCMV-neutralization studies. Antibodies to pUL131A, pUL130 and pUL128 are prime candidates for the differential neutralizing activity; this possibility was partially supported here by showing the blocking activity of anti-pUL131A/-pUL130/-pUL128 mAbs or monospecific Ig.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Cell cultures.
Three types of cell culture (of endodermic, mesodermic and ectodermic origin) were used for all of the neutralization assays: human umbilical vein endothelial cells (HUVECs), human embryonic lung fibroblasts (HELFs) and retinal pigmented epithelial (ARPE-19) cells. HUVECs were obtained by trypsin treatment of umbilical cord veins and used at passages 2–5, following testing for HCMV DNA by PCR to rule out asymptomatic congenital HCMV infection. HELFs were derived from a cell strain developed in the laboratory in 1980 and were used at passages 20–30. ARPE-19 (ATCC no. CRL-2302) is a spontaneously arising retinal pigmented epithelial (RPE) cell line derived from the human eye.
Viruses.
Four viruses were used in this study: VR1814, its deletion mutant RVFIX
UL132–128, and two variants of AD169. VR1814, originally recovered from cervical secretions of a healthy pregnant woman (Revello et al., 2001), was propagated extensively in HUVECs and has consistently been found to be competent for growth in HUVECs and leukocyte transfer (and is thus referred to as Huv+Leuk+). Propagation of VR1814 in ARPE-19 cells consistently preserved its Huv+Leuk+ characteristics. In addition, the deletion mutant RVFIXUL132–128 of the bacterial artificial chromosome (BAC)-cloned derivative of VR1814 RVFIX (Hahn et al., 2002) was propagated in HELFs as Huv–Leuk– (Hahn et al., 2004). Finally, the HCMV reference laboratory strain AD169 (originally obtained from the ATCC) was found to be Huv–Leuk–, due to a 1 nt insertion in UL131A (Revello et al., 1998, 2001; Hahn et al., 2004), yet it was shown to be able to revert the UL131A mutation to the wild-type (WT) sequence and to reacquire both properties, and thus to become Huv+Leuk+, after adaptation to growth in HUVECs (Gerna et al., 2003). In this study, the Huv–Leuk– and Huv+Leuk+ variants of AD169 have been referred to as AD169m131 and AD169WT131, respectively. HCMV strains grown in HUVECs (Huv+) were also able to grow in ARPE-19 cells and HELFs, whereas Huv– strains could only grow in HELFs. Thus, neutralization assays could be performed in HUVECs and ARPE-19 cells using Huv+ strains only, whereas in HELFs, all HCMV strains could be tested.
Microneutralization assays.
Serial dilutions (from 1 : 10 to 1 : 640 as a screening neutralization assay, and from 1 : 10 to 1 : 10 240 for neutralizing antibody end-point determination) (50 µl) of heat-inactivated human sera, as well as serial concentrations of mAbs, in minimal essential medium were incubated in triplicate for 60 min at 37 °C with an equal virus volume containing 100 p.f.u. Virus–antibody mixtures were then added to confluent cell monolayers previously seeded in 96-well microtitre plates and centrifuged at 700 g for 30 min. Following an additional 60 min incubation at 37 °C in a 5 % humidified atmosphere, inoculum was removed, cells were washed and medium supplemented with 2 % fetal calf serum was added. After 48 h incubation, cells were fixed with absolute ethanol and stained with p72-specific mAb and a Vectastain ABC kit (Vector Laboratories). The substrate was 3-amino-9-ethylcarbazole (Sigma). The serum dilution or antibody concentration inhibiting virus infectivity by 50 % or more with respect to virus controls was reported as the neutralizing-antibody titre.
Cloning, expression and purification of pUL128 and pUL130.
An open reading frame (ORF) UL128 fragment encoding mature pUL128 was amplified from a cDNA template (Hahn et al., 2004) and the amplimer was inserted into the pTrcNHisB expression vector (Invitrogen). The resulting recombinant pUL128 was expressed in Escherichia coli strain BL21 and purified by immobilized metal-affinity and ion-exchange chromatography (Patrone et al., 2007).
The plasmid pGTK-GST-UL130 core carrying a fragment of HCMV UL130 (nt 11–399), kindly provided by D. Wang and T. Shenk (Department of Molecular Biology, Princeton University, Princeton, NJ, USA), was used to express and purify pUL130 as reported previously (Wang & Shenk, 2005b). Purified pUL128 and pUL130 proteins were used for development of ELISA and Western blot assays for antibody testing. In addition, they were used for mouse immunization.
Cloning and expression of UL131A, UL130 and UL128 in HeLa and 293FT cells.
Cloning of UL130 into the pcDNA3.1(+) eukaryotic expression vector has been described previously (Patrone et al., 2005). Intron-less UL131A and UL128 ORFs were amplified from a cDNA template obtained by reverse transcription of total RNA extracted from HUVECs infected with VR1814, and cloned into pcDNA3.1(+). HeLa and 293FT (Invitrogen catalogue no. R700-07) cells were transfected with individual constructs or co-transfected with combinations of the constructs by using Lipofectin reagent (Invitrogen), according to the manufacturer's recommendations. Seventy-two hours post-transfection, cells were harvested and spotted onto glass slides by using a cytocentrifuge for the indirect fluorescent antibody (IFA) assay for antibody reactivity testing.
pUL131A, pUL130 and pUL128 antibodies.
Murine mAbs specific for pUL130 (3E3 and 3C5) and pUL128 (4B10) were a gift from T. Shenk (Department of Molecular Biology, Princeton University, Princeton, NJ, USA) and were generated by using glutathione S-transferase (GST) fusion proteins as immunogens (Wang & Shenk, 2005b). The pUL131A rabbit antiserum was raised by immunizing animals with two pUL131A peptides (Adler et al., 2006) and was a generous gift from B. Adler (Max von Pettenkofer Institut fur Virologie, Ludwig-Maximilians Universitat Munchen, Munchen, Germany). A panel of 17 pUL128 mAbs was generated in the laboratory by using the pUL128 protein obtained and purified as reported above. Multiple protein inoculations preceded the fusion step. An ELISA assay was developed to screen hybridoma supernatants and mAbs, using pUL128 as a coating protein. In parallel, an ELISA assay was developed to test antibodies to pUL130. Sera and antibodies to test for anti-pUL130 activity were assayed in parallel by an ELISA for GST antibodies. In cross-ELISA assays, no cross-reactivity was detected between mAbs to pUL128 and pUL130. Rabbit immune serum to pUL131A was tested in parallel with the relevant pre-immune serum. Following initial testing of individual mAbs, most testing was performed by using two pools of mAbs to pUL128 (4B10, D1F7 and Z9G11) and pUL130 (3C5 and 3E3). In addition, gH mAb was purchased from US Biological.
Inhibition of HCMV infectivity, plaque formation and leukocyte transfer by mAbs and human sera.
Hybridoma clones were propagated by using Celline CL1000 two-compartment bioreactor technology (Integra Biosciences AG). This technique permits high mAb yields [up to 1 mg (ml supernatant)–1]. Decreasing mAb and rabbit immune serum Ig concentrations (from 300 to 3 µg ml–1) were tested for HCMV-neutralizing activity, as well as inhibition of HCMV plaque formation and leukocyte transfer from HCMV-infected cells, either individually or in combination. The neutralizing activity was tested as reported above.
Following virus adsorption (50 f.f.u.) and centrifugation for 30 min at 700 g at room temperature, inhibition of cell-to-cell spreading was investigated by transferring infected cell monolayers to a CO2 incubator at 37 °C for 60 min. After inoculum removal and cell washing, a medium containing different antibody amounts was added. Finally, 96 h post-inoculation, 24-well microplate cell monolayers were fixed and stained by IFA using a p72-specific mAb pool as reported previously (Gerna et al., 1990). The percentage of immediate-early (IE) plaque inhibition was determined by counting the number of IE plaques in wells containing the antibody with respect to wells containing virus controls. All experiments were done in triplicate.
To perform leukocyte transfer-blocking experiments, antibodies were incubated, either individually or in combination, at 37 °C with monolayers of HCMV-infected HUVECs or HELFs 2 h prior to and overnight during co-cultivation with leukocytes (Revello et al., 1998; Gerna et al., 2000). Both types of cell culture were infected with VR1814 (propagated in HUVECs) for at least 96 h prior to co-culture. Following co-culture, leukocytes were purified by migration through a Transwell device. Control experiments were done in the absence of mAbs. Following fixation with formalin and permeabilization, leukocytes were then tested for the presence of HCMV pp65 within the nucleus by using a pool of three pp65-specific mAbs reactive with different pp65 epitopes (Gerna et al., 1992). The number of pp65-positive leukocytes per 2x105 leukocytes examined was determined. Experiments were done in triplicate for each condition. The percentage inhibition of leukocyte transfer was determined by counting the number of pp65-positive leukocytes in co-culture experiments done in the presence of antibody with respect to co-culture experiments performed in the absence of the relevant antibody.
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RESULTS |
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. In HELFs, according to the conventional assay, HCMV (VR1814)-neutralizing activity was undetectable within 10 days post-infection, then became slightly positive within 30–60 days, and reached neutralizing-antibody titre peaks of 15.7 and 12.0 within 6 months and within 1 year or more, respectively. On the contrary, when tested in HUVECs, sera from the same patients were already positive for neutralizing antibody within 10 days after onset of infection, then peaked at a level of 122.6 after 30 days, and reached peaks of 320.0 within 60 days, 545.4 at 90 days and 534.1 at 6 months. Then, the titre started dropping slowly about 1 year or more after infection. In this group of sera, the end-point dilution tested was 1 : 640. Thus, titres equal to or greater than 640 were reported as 640 (Fig. 1). Neutralizing-antibody titres similar to those obtained in HUVECs using the WT virus VR1814 were found when testing sequential sera from 10 pregnant women with primary HCMV infection in epithelial ARPE-19 cells using the same WT virus, VR1814. The previously reported deletion mutant RVFIXUL132–128 (replicating in HELFs only, and consisting of the VR1814 genome cloned in a BAC and deprived of all three genes of the locus UL131A–UL128; Hahn et al., 2004) showed neutralizing-antibody response kinetics that overlapped those of VR1814 in HELFs (Fig. 1a).
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), we observed neutralizing-antibody response kinetics comparable to those found in ARPE-19 cells and HUVECs using VR1814 (Fig. 1b
). On the other hand, the revertant AD169WT131 virus was neutralized by the same convalescent sera in HELFs at a level comparable to that of the AD169 reference laboratory-adapted strain (unable to grow in HUVECs) harbouring a frameshift UL131 mutation (AD169m131) (Fig. 1b).
When considered individually, six of ten women with primary HCMV infection became positive for neutralizing antibody against WT virus (VR1814) in HELFs at a titre of 10 more than 3 months after onset of infection, whereas all women tested early for neutralizing antibody against VR1814 in HUVECs were negative within 10 days, and positive by 10 days after onset of infection. Appearance of neutralizing antibody was generally associated with the nearly simultaneous appearance of IgG and IgM antibodies to HCMV, IgG to HCMV gB, and low IgG avidity, typical for the convalescent phase of a primary HCMV infection (Revello & Gerna, 2002). Five representative cases of primary HCMV infection, showing the neutralizing-antibody kinetics measured in both HELFs and HUVECs against either VR1814 and its deletion mutant RVFIX132–128 or AD169WT131 and AD169m131, are reported in Table 1. In these women, end-point neutralization titres were determined. In patients 4 and 5, neutralizing titres in HUVECs were consistently 2-fold higher than those in ARPE-19 cells. In addition, neutralizing titres in HELFs were <10 or low (10–40) during the entire follow-up period.
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80 (Table 1), in transplant recipients, neutralizing-antibody titres in HELFs reached levels ranging from 640 to 1280, remaining low (10–40) only in some patients, such as patient 2 (Table 2), who had a very low viral load. In patient 4 (Table 2), the difference between neutralizing-antibody titres in HELFs and HUVECs was 16-fold at day 44, becoming 2-fold in the following months, until reaching parity at day 377 after transplant. In this patient, the neutralizing activity in HUVECs was 4- to 8-fold higher in ARPE-19 cells than in HUVECs between days 58 and 377 after transplantation (Table 2). In patients 3 and 5, the difference in neutralizing-antibody titre between HELFs and HUVECs was 16-fold at the end of follow-up, whilst titres in HUVECs and ARPE cells were comparable. These different conditions observed in HCMV infections of transplant recipients reflected the patterns observed in healthy people with remote infections. In these subjects, the differential neutralizing activity observed in the two cell systems (HUVECs and HELFs) could be high, intermediate, low or absent (Table 3). Testing of a single hyperimmune globulin preparation gave a titre of 2560 in HUVECs and of 20 in HELFs.
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), whilst antibodies to pUL128 and pUL131A showed only cytoplasmic reactivity (Fig. 2a, d). IFA reactivity was tested on cells that had been paraformaldehyde-fixed and either NP-40-permeabilized or not. No cross-reactivity among antibodies with different specificities was observed. Testing of antibody reactivity was done on cells transfected with one, two or three plasmids each carrying one individual gene. Antibodies were also found to recognize the relevant protein by ELISA.
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), whereas sera from HCMV-seronegative donors did not (data not shown). Of human sera reactive by ELISA with either pUL128 or pUL130, some reacted by IFA with 293T cells transfected with UL131A, UL130 (Fig. 2f) or UL128 (Fig. 2e) plasmids, whilst some others did not (Table 4). A hyperimmune globulin preparation (pool of human Ig) was reactive by ELISA with both pUL128 and pUL130. These findings document that antibodies against these proteins are produced after natural infection.
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). When antibodies to all three viral products from this locus were used in combination, 50 % infectivity neutralization was also reached at a concentration of 15–20 µg ml–1 for each antibody, and the percentage residual infectivity decreased concomitantly with the increase in antibody concentration. Finally, when antibodies to gH were added to the combination of antibodies to pUL131A, pUL130 and pUL128 (300 µg ml–1 per antibody), complete (100 %) neutralization was reached in HUVECs with no residual infectivity, which started to appear concomitantly with the decrease in antibody concentration. In HELFs, unlike HUVECs, use of gH neutralizing antibody alone (300 µg ml–1) was sufficient to reach 100 % neutralization, whereas no effect was observed with individual or combinations of mAbs to pUL131A, pUL130 and pUL128 in the absence of mAbs to gH. Based on these data, the combination of pUL130 and pUL131A antibody appeared to display a major neutralizing effect in HUVECs, but not in HELFs (Fig. 3a).
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). In other words, in HELFs, even the highest concentrations of pUL131A, pUL130 and pUL128 antibody did not affect plaque formation. Antibody to pUL130, both in double combination with pUL128 or pUL131 and in triple combination with both pUL128 and pUL131 antibody, were able to cause 50 % plaque inhibition in HUVECs at a concentration of 30–50 µg ml–1. Conversely, plaque formation was inhibited in HELFs and HUVECs both by gH alone and by pUL131A+pUL130+pUL128+gH antibody at any antibody concentration tested (Fig. 3b).
Blocking of HCMV and HCMV product transfer from infected HUVECs, ARPE-19 cells and HELFs to leukocytes by antibodies to pUL131, pUL130, pUL128 and gH
HCMV transfer to leukocytes was shown to require the presence of a functional UL131A–128 locus in the genome of the virus strain infecting permissive cells (either HUVECs or HELFs), transferring the virus or viral products to leukocytes during cocultivation (Hahn et al., 2004; Gerna et al., 2006b). In other words, the lack of the locus of one of the three genes or even a missense mutation in one gene prevented transfer of virus and viral products to either polymorphonuclear leukocytes or monocytes. When different antibodies were assayed for blocking activity, a 50 % block of leukocyte transfer was achieved by pUL130 mAb pool at a concentration of 20 µg ml–1. No blocking effect was shown by pUL128 mAb pool or pUL131A antiserum alone (Fig. 3c). In addition, all three double-antibody combinations to pUL131A, pUL130 and pUL128 showed 50 % blocking activity at a concentration of 10–30 µg ml–1, whilst the combination of all three antibodies to pUL131A, pUL130 and pUL128, in association or not with gH-neutralizing mAb, showed the same effect at 3 µg ml–1. No substantial difference in the inhibitory effect was shown among the different antibodies, whether these were tested on HUVECs or HELFs.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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In our view, this is a significant finding for two main reasons. First, it shows that neutralizing antibodies that may inhibit replication in the majority of HCMV-permissive cell types in vivo are produced much sooner after primary infection than thought previously. Neutralizing antibodies preventing HELF infection are mostly detected late and increase in titre during reactivation episodes. It appears that, on the contrary, in primary HCMV infections, the neutralizing-antibody response is targeted to the protection from viral infection of endothelial and epithelial cells. Second, it shows that conventional neutralization assays using fibroblasts are misleading, as they fail to identify the whole spectrum of neutralizing activity.
We recently reported on the indispensability of the UL131–UL128 locus for HCMV to infect endothelial cells and to transfer virus to leukocytes (Hahn et al., 2004). Subsequently, the same finding was reported for dendritic cells (Gerna et al., 2005) and epithelial cells (Wang & Shenk, 2005a). Recently, UL131A–128 gene products have been shown to promote gB conformational transition and gB–gH interaction during the HCMV entry process in HUVECs (Patrone et al., 2007). According to these findings, when entry is allowed to initiate, gB changes to an open, protease-sensitive structure, interacting with gH and promoting fusion execution. This fusion mechanism, requiring a transient gB–gH fusion complex, is similar to that proposed for herpes simplex virus 1, in which a conformational change of gD promotes the sequential recruitment of gB and gH–gL in a transient complex that disassembles after fusion (Gianni et al., 2006; Subramanian & Geraghty, 2007). Thus, UL131A–128 gene products would be fusion activators and possibly entry receptor ligands. Most recently, it was shown that pUL128, pUL130 and pUL131A must all bind simultaneously onto gH–gL for the production of complexes that are incorporated into the virion envelope and can function in entry into epithelial and endothelial cells (Ryckman et al., 2008).
These distinctive mechanisms of virus entry into HUVECs and other cell types as opposed to HELFs, requiring the pUL131A–pUL130–pUL128 complex or not, respectively, support a pUL131A–pUL130–pUL128 complex-dependent Ig reactivity as the possible molecular basis for the differential HCMV-neutralizing activity of human sera. According to this hypothesis, antibodies that either bind pUL131A–pUL128 proteins directly, or depend on their incorporation in the HCMV envelope for recognition of other envelope proteins, are essential for neutralization. This neutralizing component would be ineffective against both WT virus and the UL131A–UL128-negative strains in the HELF system, in which the pUL131A–pUL130–pUL128–gH–gL complex is irrelevant for entry. On the contrary, in HUVECs and the other cells in which the complex is required for entry, the neutralizing component would display its effects.
In the second part of this work, we report a few observations that corroborate the direct neutralization of the pUL131A–pUL130–pUL128 complex as the mechanism. First, ELISA or IFA showed that neutralizing human sera do contain antibodies to UL131A–128 gene products. Second, a set of murine monoclonal and rabbit polyclonal antibodies raised against pUL128, pUL130 and pUL131A was tested. Their specificity was documented by the IFA, Western blot and ELISA recognition of the relevant gene products in cells transfected with constructs carrying the single genes or the entire UL131A–UL128 locus. Importantly, in three assay formats in which the neutralization of HUVEC infection, plaque formation in HUVECs and virus transfer to leukocytes were tested, monospecific antibodies were assayed either individually or in combination. In the past, 50 % neutralization was observed by using a single antibody, such as anti-pUL130 mAb 3E3 (Wang & Shenk, 2005b) or anti-pUL131A rabbit antiserum (Adler et al., 2006). However, in the former study, a conventional plaque-reduction assay was used, whilst in the latter study, a much higher antibody concentration than that in our study was used. By using pools of the serially diluted Ig against the three gene products, the following results were obtained: (i) 50 % neutralization of VR1814 in HUVECs was found at a concentration of 15–20 µg ml–1, regardless of whether a gH mAb was also present; in HELFs, conversely, the antibody pool to pUL131A, pUL130 and pUL128 did not display any neutralizing activity against VR1814, whereas gH mAb alone or gH mAb plus pUL131A, pUL130 and pUL128 antibodies showed a 50 % neutralizing activity at 2–5 µg ml–1; (ii) the antibody pool to pUL131A, pUL130 and pUL128 displayed a linear dose–effect relationship with respect to plaque-formation inhibition, reaching 50 % inhibition at a concentration of 30 µg ml–1; (iii) finally, the antibody pool to pUL131A, pUL130 and pUL128 showed a dose–effect relationship when the block of virus transfer from HCMV-infected HUVECs or HELFs to leukocytes was assayed, reaching a 50 % leukocyte-transfer block at a concentration of 3–5 µg ml–1. This in vitro analysis additionally suggested a prominent role of pUL130 among the pUL131A–pUL130–pUL128 complex because, in all three assay formats, the anti-pUL130 immunoglobulins had to be present for a significant activity to be observed. Furthermore, anti-pUL130 alone could reduce virus transfer to leukocytes significantly. Surprisingly, this is the only biological function (among those studied) requiring the intervention of UL131A–128 gene products for virus transfer to leukocytes, not only from infected HUVECs, but also from infected HELFs. This finding suggests that virus transfer to leukocytes from HELFs, unlike virus neutralization and plaque inhibition, requires the intervention of the gene products of the UL131A–128 locus.
Additionally, the protective effect of neutralizing antibodies in vivo appears difficult to understand, given the infrequent release of cell-free virus in blood or organic fluids, unless indirect mechanisms of action, such as antibody-dependent cellular cytotoxicity, are considered. As far as our results with animal antibodies can be extrapolated to human infection, they underpin two additional pathways of protection from HCMV pathogenicity by pUL131A, pUL130 and pUL128 antibodies. One is blocking of cell-to-cell spreading, which applies to virus dissemination inside an organ tissue; the other involves blocking of bidirectional virus transfer between infected endothelial cells of blood vessels and peripheral blood leukocytes.
The above pieces of evidence, although in agreement with the purported role of anti-pUL131A/-pUL130/-pUL128 antibodies in human serum neutralizing activity, do not prove it directly. A direct proof would require subtracting the neutralizing activity by adsorption of human sera with pUL131A, pUL130 and pUL128. We indeed tested sera following adsorption with purified recombinant proteins pUL128 and pUL130 (recombinant pUL131A was not available in soluble form). This treatment did ablate serum reactivity in ELISA to both antigens, yet it did not reduce the neutralizing activity in HUVECs significantly (data not shown). It is quite possible that improper folding of proteins expressed in a prokaryotic system and/or the absence of a complete and properly assembled gH complex fail to reproduce the pUL131A/pUL130/pUL128 epitopes crucial for neutralization. Alternatively, it is also possible that inclusion of the pUL131A–pUL130–pUL128 complex in the viral particle may expose different epitopes on glycoproteins, such as gH and gB, that are the target of neutralizing antibody. Resolving this point will be important, particularly for the design of vaccines.
As a consequence of this study, it seems advisable to recommend that the neutralizing activity of human sera be determined in endothelial or epithelial cells, leaving the neutralization titre measured in HELFs as a secondary end point to be considered for comparison. Moreover, administration of Ig preparations has been proposed repeatedly for the prevention and therapy of HCMV infection and disease in both immunocompetent and immunocompromised patients (Bass et al., 1993; Messori et al., 1994; Andreoni et al., 2001; Nigro et al., 2005); neutralizing human mAbs have been developed for the same purpose (Boeckh et al., 2001). Again, our study suggests that a complete evaluation of the protective activity of Ig preparations/human mAbs must include an assay in HUVECs or ARPE-19 cells, and that anti-pUL131A, -pUL130 and -pUL128 human mAbs may be a valuable component in the formulation of such immunodrugs.
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ACKNOWLEDGEMENTS |
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Received 16 October 2007;
accepted 12 December 2007.
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), HELFs (
) and ARPE-19 cells (
), and against its deletion mutant RVFIX
). (b) Serum neutralizing activity against AD169 with UL131A mutation in HELFs (
