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In SCID-hu Mice, Passive Transfer of a Humanized Antibody Prevents Infection and Atrophic Change of Medulla in Human Thymic Implant due to Intravenous Inoculation of Primary HIV-1 Isolate¹

Yukari Okamoto^*, Yasuyuki Eda, Atsuo Ogura, Shinwa Shibata, Takashi Amagai^§, Yoshimoto Katsura^¶, Toshihiko Asano, Kazuhiko Kimachi, Keiichi Makizumi and Mitsuo Honda^2,*

^* AIDS Research Center, National Institute of Infectious Diseases, Tokyo; Chemo-Sero-Therapeutic Research Institute, Kyokushi Kikuchi, Kumamoto; Department of Veterinary Science, National Institute of Infectious Diseases, Tokyo, Japan; ^§ Department of Immunology and Microbiology, Meiji College of Oriental Medicine, Kyoto; and ^¶ Department of Immunology, Institute of Chest Diseases, Kyoto University, Kyoto, Japan

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Using SCID-hu mice, it was tested whether humanized mAb Rµ5.5 could prevent infection by HIV-1 i.v. inoculation. The Ab that recognizes the IHIGPGRAFYT motif in the principal neutralizing determinant (PND) of HIV_MN, as well as the original mouse mAb µ5.5, neutralized HIV_MN with high activity. Seven primary field isolates from Japanese hemophiliacs seropositive for HIV-1 clade B were compared for their reactivities to Rµ5.5. Rµ5.5 was effective, particularly against the viruses that matched amino acid sequences of the PND region of HIV-1, and it completely neutralized primary isolates. Moreover, the passive transfer of the Ab elicited protection against challenge by the primary isolates in SCID-hu or hu-PBL-SCID mice after i.v. inoculation with the virus by both quantitative PCR and PBMC-based virus isolation in vitro. Further, inoculation with the Ab also prevented the atrophic change in the medulla of the thymic transplant that was induced by i.v. inoculation of the virus. Thus, the humanized neutralizing Ab Rµ5.5 appears to protect SCID-hu mice from infection by primary field isolates.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The principal neutralizing determinant (PND)³ of the V3 region of the surface glycoprotein gp120 of HIV-1 is an immunodominant portion of this protein responsible for neutralizing both humoral immune responses and cellular immune responses against HIV-1 (1, 2, 3, 4). Anti-PND responses are elicited both in naturally infected humans (5, 6) and in animals infected experimentally with HIV-1 (7). Because of the importance of the PND region as a neutralization epitope, this region of the V3 loop has been one of the major targets in the development of anti-HIV agents (8, 9) and in serologic analysis of the clinical status of individuals with HIV-1 (6).

Recently, chemokine-mediated suppressions of HIV-1 infection have been reported, and their chemokine receptors have been demonstrated to act as HIV cofactors (10, 11). Interestingly, the V3 region of the gp120 envelope protein was demonstrated to be a critical determinant for susceptibility to the chemokine-mediated suppression of HIV-1 (12, 13), suggesting that anti-V3 Ab may neutralize HIV-1 infection by blocking the binding of HIV-1 to the coreceptors on the surface of the target cells. Although HIV-1 has been known to be neutralized by various Abs including anti-PND Abs directed against its envelope protein (1, 2, 3, 14), primary isolates of HIV-1 have been reported to be relatively resistant to neutralization by mAbs to gp120 (15). However, primary isolates may not be intrinsically resistant to neutralization by anti-V3 mAbs in that a human mAb 447-52D was found effective (15, 16).

In this study, we reshaped the anti-PND mAb µ5.5 (17) and established a humanized Ab, Rµ5.5, with very potent neutralizing activity. Further, we studied the antiviral characteristics of the humanized Ab against various primary isolates of HIV-1. In addition, passive transfer of the Rµ5.5 Ab was performed with primary HIV-1 isolates in thymus/liver-transplanted (Thy/Liv) SCID-human (hu) (18, 19) or human peripheral leukocyte-reconstituted SCID (hu-PBL-SCID) mice (20), because both Thy/Liv SCID-hu and hu-PBL-SCID mice seem to be convenient animal models for evaluating the potency of prophylactic agents to prevent the infection by primary clinical isolates. It was also noted that passive transfer of Rµ5.5 protects against the atrophy within the thymic medulla that is seen when the mouse is infected by a virus alone.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Study subjects

Seven individuals seropositive for HIV-1 were selected from Japanese hemophiliacs between 21 and 39 years of age. HIV infection was detected by routine anti-HIV Ab ELISA and Western blotting and confirmed by virus isolation at the National Institute of Infectious Diseases, Tokyo, Japan (9, 21).

Mice

Inbred CB-17 scid/scid (SCID) mice maintained at the National Institute of Infectious Diseases, Japan, were used in this study. For construction of SCID-hu mice, human thymus and liver tissues from fetuses (HIV-free proven) of 17 gestational weeks were implanted under the left kidney capsule of Thy/Liv SCID-hu mice at the age of 7 to 15 wk (9, 22). After 3 mo of feeding, aliquots of the mice were sacrificed to confirm the growth of the tissues transplanted by surgery. In each of the mice tested, the grafts had increased in size to more than 5 x 5 x 4 mm, and the expected CD45-positive thymocyte subpopulations were confirmed by flow cytometry. The remaining mice were subjected to the following experiments. The mice were used for experiments after three mo of feeding. Hu-PBL-SCID mice were reconstituted by i.p. injection of 2 x 10⁷ freshly isolated normal human PBL suspended in 0.5 ml of PBS. Two weeks after PBL injection, only mice confirmed to be reconstituted with human PBLs were used for HIV-1 injection (9). The efficacy of the humanization of the PBL-SCID mice was determined by measuring the serum levels of human Ig in the mice.

Isolation and sequencing of µ5.5 mAb V region gene

Mouse mAb µ5.5 was selected as a neutralizing Ab against HIV_MN as described above (17). µ5.5 is made by conventional method for mAb production using HTLV-III MN Ag with BALB/c mice, and the isotype is mouse IgG1() (Y. Eda, manuscript in preparation, Chemo-Sero-Therapeutic Research Institute, Kyokushi Kikuchi, Kumamoto, Japan). RNA was extracted from µ5.5 hybridomas in accordance with the conventional method, and first-strand cDNA was synthesized using a cDNA Synthesizer System Plus (Amersham, Buckinghamshire, U.K.). The 5' primers of mouse Ig V regions and the 3' primers of the J region were designed based on the nucleotide sequence database of mouse Ig as classified by Kabat et al. (23). The V region primers contained HindIII sites, and the J region primers contained BamHI sites. PCR was performed using the first-strand DNA as a template and the V and J region primers. The PCR products were cloned into the HincII site of pUC18. Sequencing of the V region gene in pUC18 was carried out using Sequenase (Amersham).

Preparation of reshaped human µ5.5 mAb (Rµ5.5)

The transplantation of complementarity determining regions (CDRs) and a part of framework regions (FRs) of µ5.5 into human V regions was carried out in accordance with the method for preparing a reshaped Ab, as previously described (24). In brief, CDRs and a part of the FRs of the V_H regions of µ5.5 were transplanted into the V_H region having a FR region of human subgroup I (SGI), whereas CDRs of the V_L region of µ5.5 were transplanted into the V_L region having a FR region of human -chain REI (V_L) (25). Mutagenesis oligonucleotide primers coding for the portion of V_H or V_L region of µ5.5 to be transplanted plus the flanking portion were used to hybridize to the FRs of human V region and were annealed to the V region gene of SGI or REI in single-strand M13mp18 DNA. The template M13 DNA was cleaved with NciI and was digested with exonuclease III to give only the mutated M13 DNA. Then, PCR was carried out using the product after exonuclease III digestion as a template along with a universal primer that contains a sequence complementary to the 5' site of M13mp18 and a reverse primer which contains the same sequence as the 3' site of M13mp18. The PCR products were digested with BamHI and HindIII, and were inserted into the BamHI-HindIII site of pUC18. DH5 (Life Technologies, Gaithersburg, MD) was used for transfection of these plasmids. As a primary screening, a colony was hybridized by using the mutagenesis primers, to select clones with successful mutagenesis. Then, as a secondary screening, a plasmid was prepared from the clones obtained in the primary screening, and a sequence was carried out with Sequenase (Amersham) to confirm correct transplantation. The V_H and V_L fragments were termed RHµ5.5 and RLµ5.5, respectively; they were digested with HindIII and BamHI, and inserted into the HindIII-BamHI site of expression vectors human cytomegalovirus (HCMV)-1 or HCMV-. A product of these reshaped µ5.5 Ab plasmids was examined in a transient expression system using COS7 cells (ATCC CRL 1651; American Type Culture Collection, Rockville, MD).

A mixture of RHµ5.5 and RLµ5.5 plasmids was then transfected into SP2/0-P3X63Ag8.653 hybrid mouse myeloma cell line SFT (provided by Mr. K. Nishiyama, Chemo-Sero-Therapeutic Research Institute). Stable transformants were screened by resistance to G418 (Life Technologies). Finally, a clone Rµ5.5/SFT, which produced a high amount of Rµ5.5 mAb, was selected, by using ELISA, to measure the binding of the Ab to SP1 synthetic peptide YNKRKRIHIGPHRAFYTTKNIIG. After a large scale culture of the Rµ5.5/SFT cell, the Rµ5.5 mAb was purified from the culture supernatant by protein A-Sepharose affinity chromatography.

Epitope mapping of Rµ5.5

The epitope mapping was performed using an Epitope Scanning Kit (Chiron Mimotopes, Pty, Victoria, Australia). Briefly, overlapping peptides from the region of amino acids 306 to 325 of the HIV_MN gp120 envelope were synthesized using an Epitope Scanning Kit. The reactivity of each peptide overlapping with Rµ5.5 mAb was examined by ELISA.

Preparation of clinical HIV isolates and sequencing of their V3 loops

Viral stocks of HIV-1 primary clinical isolates were prepared by coculturing PHA-activated human PBMC from both HIV-seropositive and normal individuals as described by Gorny et al. (16). The cell-free supernatant was stored at -130°C until used as the virus source. Characteristics of the syncytium formation of the isolates were examined using MT-2 cells.

The supernatant virions were precipitated by ultracentrifugation (45,000 x g), the virion RNA was extracted, reverse-transcribed to DNA by using an OD3 primer (nucleotide 7345 to 7369 = 5'-AAATTCCCCTCACAATTAAAACTG-3'), and the DNA of the V3 loop in the HIVenv region was sequenced as described (9). The positions of the oligonucleotides are numbered relative to the HXB2 isolate in the ENTREZ database (National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD).

Virus neutralization assay

The classical MT4 cell-based virus neutralization assay was performed to screen the neutralization Ab for HIV-1 (1). Briefly, the neutralization of cell-free HIV infection against MT-4 cells by the Abs was determined as follows. Fifty microliters of 200 TCID₅₀ per milliliter of HIV_MN or HIV_RF was mixed with an equal amount of serially diluted µ5.5 mAb, Rµ5.5 mAb, or 0.5ß mAb (1, kindly provided by Dr. S. Matsushita, Department of Internal Medicine, Kumamoto Medical School, Kumamoto, Japan). After 1 h of incubation at 37°C, 100 µl of 1 x 10⁴ MT-4 cells were added. The plate was then incubated at 37°C in 5% CO₂ atmosphere for 4 days, and the number of syncytium cells was counted using an inverted microscope. Each 5 x 10³ cells of H9/MN, CEM/LAV, or U937/RF was preincubated in 96-well microtiter plates with various concentrations of purified µ5.5 mAb, Rµ5.5 mAb, or 0.5ß mAb in 100 µl of RPMI 1640 supplemented with 10% FCS. After 1 h of incubation at 37°C, 100 µl of 5 x 10⁴ MT-4 cells were added and cultured for 18 h, and the culture was then examined for the number of syncytium cells using an inverted microscope.

The PBMC-based virus neutralization assay was performed as described (9). In brief, the diluted Abs were incubated with 20 TCID₅₀ U of HIV_MN (H9/HTLV-III_MN, AIDS Research and Reference Reagent Program, National Institutes of Health, Rockville, MD) or various clinical isolates for 60 min at 37°C, and the mixture was shaken with 2 x 10⁶ PHA-activated normal PBMC for 60 min in a 37°C water bath. After being washed, the cells were cultured in the presence of human rIL-2 (40 U/ml, Shionogi and Co., Osaka, Japan) for 7 days. The amount of HIV was measured by p24 Ag ELISA (Dinabot, Tokyo, Japan).

The in vitro neutralization activity of the Abs against field primary isolates from Japanese hemophiliacs was expressed as percentage of inhibition of p24 Ag production in the culture supernatants compared with that in the cultures to which human IgG1 from myeloma plasma or mouse plasma IgG1 was added. Virus stocks were titrated on the PHA-activated normal PBMC, and the TCID₅₀ of each virus was determined.

Passive transfer of Abs to SCID-hu or hu-PBL-SCID mice followed by virus inoculation and in vitro virus isolation

Twenty-four Thy/Liv SCID-hu mice were used per challenge virus and divided into two groups. In the first group, each mouse was given an i.p. injection of 400 µg of Rµ5.5 (26), was administered an i.v. inoculation of 1000 TCID₅₀ of HIV_MN or 100 TCID₅₀ of HIV-1 field isolates, and then fed for 3 more wk. Mice in the other group were injected with the same amount of human myeloma IgG1 followed by the same combinations of viruses and feeding. Similarly, 24 hu-PBL-SCID mice were used for evaluation of the ability of protection against the virus with 84-h challenge of the viruses. Both the Thy/Liv SCID-hu mice and hu-PBL-SCID mice that received i.v. injection of saline were used as control. All procedures for infection and maintenance of animals were performed in a biosafety level 3 facility at the National Institute of Health, Japan.

Mononuclear cell fractions of venous blood, peritoneal lavage cells, and grown human thymic transplant cells were obtained and isolated from SCID-hu mice. Mononuclear cells from the thymic transplant of Thy/Liv SCID-hu mice or from hu-PBL-SCID mice were cocultured with PHA-stimulated normal human PBMC for virus isolation as described (21). The amount of HIV in the supernatant was measured by HIV-1 p24 Ag ELISA (Dinabot).

Quantitation of HIV-1 by DNA-PCR

Total DNA was prepared from the mononuclear cells from the SCID-hu or hu-PBL-SCID mice for PCR amplification. The primer pair SK38/39 was used, and amplification was carried out for 35 cycles in a DNA thermal cycler (Perkin-Elmer Cetus, Norwalk, CT) by a modified method of Poznansky et al. (27). As a control of the PCR of genomic DNA, a 548-base pair fragment of human ß-actin DNA was amplified by the PCR method using a ß-actin primer pair (Clontech Laboratories, Palo Alto, CA). A standard curve was generated with the genomic DNA from the 8E.5 cell line cell, which contains one provirus per cell (AIDS Research and Reference Reagent Program, National Institutes of Health, Rockville, MD).

Histologic study

The Thy/Liv SCID-hu mice were sacrificed 3 wk after inoculation with HIV-1, and the grown human thymic tissue was dissected. For conventional light microscopy, tissue sections were stained with hematoxylin and eosin. Dissected thymic tissues were fixed with buffered formalin for several days at 4°C, dehydrated in ethanol, and embedded in paraffin. Some sections were stained for HIV-1 by immunofluorescence technique with a rat polyclonal Ab for HIV gp120 (Advanced Biotechnologies, Columbia, MD), followed by a fluorescein-labeled goat anti-rat IgG Ab (Organon Teknika Corp., Durham, NC) as previously described (24). Sections that were not exposed to the primary Ab were used as negative control. For identification of thymic epithelial cells in the implants, part of the specimens were doubly stained with a rabbit anti-cytokeratin polyclonal Ab (Biomeda Corp., Foster City, CA) as the primary Ab and a rhodamine-labeled goat anti-rabbit IgG Ab (Tago, Burlingame, CA) as the secondary Ab, together with the Ab described above.

Statistical analysis

Calculations of the geometric mean ± SD were carried out with a microcomputer. Significance was defined as p < 0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Production of reshaped human Ab Rµ5.5

Preliminary studies to select a potent neutralizing Ab against HIV-1 revealed that one of the hybridomas tested was designated µ5.5, which secreted the IgG1()-type mAb that neutralized against HIV_MN (17).

The stable reshaping Ab clone Rµ5.5 was produced by reshaping the V region gene fragments from anti-PND Ab producer clone µ5.5 cells. The amino acid sequences deduced from the nucleotide sequences are shown in Figure 1. The nucleotide sequences of µ5.5 V_H and V_L regions exhibited a rearrangement specific for the V region gene and showed an open reading frame that allows for expression.

View larger version (30K): [in this window] [in a new window]   FIGURE 1. Amino acid sequences of mouse, human, and reshaped human VH (A) and VL (B). The FR and the CDR segments are shown. CDR assignments are according to Kabat et al. (23). In the reshaped human V regions, the amino acids that were transferred from the mouse Ab are underlined. For the human V regions, only amino acid sequences in the FRs are shown. Human SGI VH and REI VL are the same sequences as used by Maeda et al. (24).

Neutralizing property of µ5.5 and Rµ5.5

Neutralizing activities of µ5.5 and Rµ5.5 were assessed by two different MT-4 cell-based assays. Initially, the neutralizing activity against cell-free virions was determined (Table I). Serial dilutions of µ5.5, Rµ5.5, 0.5ß or irrelevant IgGs were preincubated with the virus for 60 min at 37°C, and the mixture was poured onto susceptible MT-4 cells. After 4 days culture, syncytium cells were counted by inverted microscope. Both µ5.5 and Rµ5.5 neutralized the infection of HIV_MN. The neutralizing titer of Rµ5.5 was 0.5 µg/ml, which was higher than that of µ5.5. In contrast, the neutralization activities against HIV_IIIB/LAV isolates were not observed with the Ab. The 90% neutralization activity of Rµ5.5 against HIV_MN was 0.1 µg/ml of the Ab by PMBC-based virus neutralization assay (Fig. 2).

View larger version (17K): [in this window] [in a new window]   FIGURE 2. Humanized Rµ5.5 neutralizes HIVMN by PBMC-based virus neutralization assay. µ5.5 (), Rµ5.5 (•), or human IgG1 () Ab was incubated with the virus for 1 h at 37°C. The results are expressed as percentage inhibition, compared with control activity, and the mean of four different assays.

Epitope mapping of Rµ5.5

To define the epitope recognized by Rµ5.5, the ability of the Ab to bind to peptides was tested using a set of overlapping peptides from the region of amino acid 306 to 325 of the HIV_MN gp120 envelope protein. Each peptide was offset from its neighbor by one amino acid. Figure 3 shows that Rµ5.5 reacted with peptides longer than 11 mer that contain the IHIGPGRAFYT motif, suggesting that the binding epitope of Rµ5.5 was represented as IHIGPGRAFYT.

View larger version (23K): [in this window] [in a new window]   FIGURE 3. ELISA reactivity of reshaped human Ab Rµ5.5 with the overlapping peptides in the region covering amino acid position 306 to 325 of the HIVMN gp120 envelope. Each peptide was offset from its neighbor by one amino acid. The reactivity with purified Rµ5.5 mAb is shown on the ordinate. OD, 450 nm.

Seven primary clinical isolates, from JCI-1 to JCI-7, were obtained from Japanese hemophiliacs seropositive for HIV-1 clade B. These consisted of three isolates that possessed the same IHIGPGRAFYT sequence as HIV_MN at the neutralization epitope of the HIV-PND, and another four viruses containing one or two different sequences in the neutralization epitope (Table II). Five of the seven isolates formed syncytia in MT-2 cells compared with the SI virus of HIV_MN (Table II). Other characteristics of the isolates are also shown in Table II, and the isolates were used as virus sources.

Although HIV-JCI-1, -2, and -3 all showed the same core sequences when compared with the V3 sequence of HIV_MN, HIV-JCI-1 and -2 were strongly neutralized by Rµ5.5, which was also shown to neutralize HIV_MN. The neutralization end point at 90% or greater was from 1 to 10 µg/ml in the IgG (Fig. 4). However, HIV-JCI-3 was significantly less neutralized by Rµ5.5, suggesting that some mutation outside the binding core epitope of the HIV-PND might be responsible for binding the Ab. Normal human serum IgG did not show neutralization activity (data not shown). HIV-JCI-4, -5, -6 each had at least one mutation in the lower half of the core epitope and showed weak neutralization activity against Rµ5.5. However, the mutated upper region of the core epitope from [IHI] to [IQI] of HIV-JCI-7 was not neutralized by Rµ5.5 (Table II and Fig. 4). Thus, we demonstrated that the humanized Rµ5.5 Ab has strong neutralizing activity against clinical isolates that match the neutralization sequence motif in vitro.

View larger version (35K): [in this window] [in a new window]   FIGURE 4. Neutralization of primary field isolates of HIV-1 and HIVMN by humanized Ab Rµ5.5. The percentage inhibition of HIV production was expressed by using normal mouse IgG as a control in the PBMC-based virus neutralization assay. The results are expressed as the mean of four different assays.

Since Rµ5.5 was identified as a humanized Ab with potent neutralizing activity against primary clinical isolates, the Ab has been used as a prophylactic agent against infection by the primary isolates to both Thy/Liv SCID-hu mice and hu-PBL-SCID mice as shown in Table III and Figure 5.

View larger version (18K): [in this window] [in a new window]   FIGURE 5. The protective effect of the neutralizing Ab against HIV-1 challenge in Thy/Liv SCID-hu and hu-PBL-SCID mice. Twelve Thy/Liv SCID-hu and 12 hu-PBL-SCID mice were divided into two groups, and six mice in each group were inoculated with 400 µg/ml of Rµ5.5 or relevant human IgG1 as control IgG. The virus released from the PBMC of the hu-PBL-SCID mice or teased out of implanted thymocytes from Thy/Liv SCID-hu mice was assayed by the in vitro virus proliferation method.

By using the hu-PBL-SCID mice HIV model, HIV infection was also suppressed when Rµ5.5 was injected into the mice before the primary HIV-JCI-1 and -2 infection, as well as the effect of the Ab in HIV_MN infection by PCR analysis (Table III). In addition to the suppressive effect of p24 Ag production in the virus assay in the SCID-hu HIV model, Rµ5.5 also suppressed the production of HIV-JCI-1 in the hu-PBL-SCID mice HIV model (Fig. 5, right panel).

Thus, the protective property of the Ab against the challenge infection of primary clinical HIV-1 isolates was detected by transferring the Rµ5.5 Ab to hu-PBL-SCID mice as well as to SCID-hu mice.

Histologic study of the implanted thymus from SCID-hu mice

The thymus tissue implanted 3 wk after infection with primary HIV isolate showed an atrophic change of medullary tissue, with an involuted appearance and no cortex-medulla distinction (Fig. 6b), when compared with the tissue of the control mice not inoculated with the virus (Fig. 6a). Many Hassall’s corpuscles, found in the parenchymal region, often exhibited end-stage morphology consisting of large keratinized centers. However, the number of Hassall’s corpuscles did not seem to change significantly as compared with that of the control mice. Immunofluorescent study demonstrated that Hassall’s corpuscles were most intensively stained for the viral Ag (gp120) in the implants. Hassall’s corpuscles showed granular staining patterns not only within the cytoplasm of the crescent-shaped epithelial cells but also in the narrow intercellular spaces. A few solitary round cells positive for the virus Ag were also detected in the parenchymal regions. At least part of the HIV-positive cells in the perivascular areas were negative for cytokeratines, these most likely being thymocytes (data not shown).

View larger version (131K): [in this window] [in a new window]   FIGURE 6. Low power light microscopic observation of the thymic transplant from the Thy/Liv SCID-hu mice 3 wk after inoculation of 100 TCID of HIV-JCI-1. Hematoxylin-eosin stain, x70. a, Thymic transplant tissue after 3-mo transplantation of fetal thymus and liver. b, Thymic transplant tissue 3 wk after HIV-1 inoculation to the Thy/Liv SCID-hu mice. Atrophic tissue and Hassall’s corpuscles of varying sizes are found. The medulla is narrow, and the boundary between the cortex and the medulla has disappeared. c and d, Protection of the atrophic thymic changes in Thy/Liv SCID-hu mice transferred with the Ab Rµ5.5.

These pathologic changes that appeared after viral challenge were not seen in the graft following the administration of 400 µg of Rµ5.5 Ab (Fig. 6, c and d) or irrelevant human IgG1 (data not shown).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
This report describes our finding that the humanized Ab to the PND region of V3 loop of HIV-1 gp120, designated Rµ5.5, neutralized primary HIV-1 viruses isolated from Japanese hemophiliacs with HIV-1 clade B. Further, this initial observation was extended to find that the Ab protected both Thy/Liv SCID-hu and hu-PBL-SCID mice from infection with the primary HIV-1 strains. We also addressed the prevention of atrophy of the medulla of the engrafted thymus by administration of the Ab to mice challenged with the viruses.

The Rµ5.5 neutralization of laboratory HIV-1 strain showed strain specificity for HIV_MN. Further, the mapping of this Ab with various V3 peptides indicated that the Rµ5.5 epitope spans the IHIGPGRAFYT motif in PND in the V3 loop of HIV_MN. The neutralizing epitope at the tip of the V3 loop was also recognized in primary HIV-1 clinical isolates, which matched the amino acid sequence of PND in HIV_MN. The ability of the virus to be neutralized correlated with the expression of the sequence of the epitope. When the sequence of the epitope matched completely, the neutralization activity of the Rµ5.5 was highly potent by PBMC-based neutralization assay for clinical viruses. However, when there was substitution of the amino acid in the neutralization sequences of primary isolates, the neutralization ability was decreased. Further, Rµ5.5 failed to neutralize the viruses that have a glutamine residue at position 15 of V3 loop, suggesting that position 15 in the V3 domain of gp120 envelope glycoprotein seems to be critical for Rµ5.5-mediated neutralization against infection by primary clinical isolates.

We previously succeeded in constructing humanized Abs from 0.5ß mAb that neutralized HIV_IIIB (24). In constructing humanized Ab R0.5ß, the CDR3 gene used was from Kabat’s V subgroup III, and only a 96th amino acid was different from that of CDR3 of µ5.5. In contrast, the CDRs of µ5.5 V_H were completely different from 0.5ß V_H (24). Thus, the V_H region of the reshaped neutralizing Ab against the HIV-PND epitope suggests that it is critical for serving the immunotype-specificity of the Ab. By construction of the humanized Ab, the neutralizing activity of the reshaped Ab Rµ5.5 was estimated to be more than twofold higher, by virus neutralization assays, than that of the original mouse mAb µ5.5. These results indicate that the Ag-binding property of µ5.5 has been successfully introduced into the humanized Ab. How-ever, some of the humanized Abs reduced the Ab efficacies (23, 28).

The neutralization motif in PND of the V3 loop was identical with the consensus motif of HIV-1 clade B viruses, which are prevalent in North American and Western European countries (29). Further, the sequence was also identical with the consensus HIV-1 prevalent in Japanese hemophiliacs seropositive for HIV-1 (30, 6). Recently, we reported the detection of a type-specific Ab to the PND of HIV_MN that was highly homologous with the consensus motif of HIV-1 clade B viruses in the serum of HIV-infected Japanese hemophiliacs. Further, the Ab titer in rapid progressors was significantly lower than that of slow progressors in HIV infection. In the slow progressors, the Ab response was relatively conserved in specificity as compared to the reactivity in rapid progressors. We suggested that one of the factors that might control the course of infection could be that some anti-PND Abs neutralize the circulating free viruses in vivo. Taken together, neutralizing Abs against the PND of the V3 loop of prevalent field HIV-1 possibly limit the degree of disease progression in HIV-1 infection.

In support of the concept of neutralization activity by humoral Abs against clinical isolates of HIV-1, human mAb 447-52D (16, 31), which binds PND in the V3 loop of HIV-1, mAb 697-D (16), which binds the V2 region of HIV-1, and polyclonal serum Ab have been shown by PBMC-based virus neutralization assay to neutralize primary clinical isolates. Furthermore, serum Ab against HIV gp120 has been reported by resting cell assay (32) to neutralize primary clinical isolates. Those studies, however, did not address the protective property of the Abs against HIV-1 challenge. In this report, humanized mAb Rµ5.5, which recognized the PND of HIV-1, induced potent neutralizing activity against clinical HIV-1 clade B. Furthermore, the Ab blocked the infections of clinical isolates of HIV-1 by passive immunization of the Ab in SCID-hu HIV models. The passive immunization by the anti-HIV candidate Abs seems to be beneficial for studying their protective property against various clinical isolates in blocking HIV-1 infection in SCID-hu HIV models.

In addition, we showed that the primary isolates of HIV-1 clade B induce an atrophic change in the medulla in engrafted thymic tissue in the Thy/Liv SCID-hu mice model for HIV-1 by i.v. challenge of the virus. Interestingly, Rµ5.5 was also characterized by its protective property against the pathologic changes of the engraft by the passive transfer of candidate neutralization Ab in the Thy/Liv SCID-hu mice model for HIV-1. Previously, we reported on distribution patterns of HIV-1 within the human thymus in Thy/Liv SCID-hu mice that had been infected with HIV-1 by a single i.v. injection of virus; and we also reported that the viral Ags were demonstrated predominantly in Hassall’s corpuscles, by immunofluorescence studies and electron microscopy (22). In these reports, we extended the study using the clinical isolates as the virus source. Also, the virus detections were observed mainly in Hassall’s corpuscles and somewhat in surrounding thymocytes or in round thymic epithelial cells. In this atrophic region of the medullary tissue, boundaries between the cortex and medulla were obscure and apoptotic cells were found (T. Sata et al., unpublished observation, Laboratory of Pathology, AIDS Research Center, National Institute of Health, Japan).

We showed that the passive protection strategy could possibly be used clinically. The neutralization activity of the µ5.5 mAb was very high, and epitope specificity of the protective effect of the mAb was highly restricted to the PND sequences of HIV-1. The sequence matching rate of the binding epitope of the Ab with clinical isolates in Japan is about 12% (6), suggesting that different mAbs would be necessary to neutralize all the primary isolates in Clade B HIV in Japan. Broadly neutralizing anti-V3 mAbs will probably be better candidates for the passive protection strategy to control the HIV-1 infection. Our results suggest that passive immunization with appropriate Abs with potent protective activity against clinical isolates of HIV-1 could be considered for the prophylaxis of HIV-1 infection.

	Acknowledgments

 
We thank Drs. H. Yoshikura (AIDS Research Center, National Institute of Infectious Diseases, Tokyo, Japan), S. Yamazaki (National Institute of Infectious Diseases, Tokyo), R. C. Krause (Fogarty International Center, Bethesda, MD), and Hiroaki Maeda (Chemo-Sero-Therapeutic Research Institute, Kyokushi Kikuchi, Kumamoto, Japan) for their helpful discussions.

	Footnotes

 
¹ This work was supported by a grant-in-aid from the Ministry of Health and Welfare, Japan, the Program for the Promotion of Fundamental Studies in Health Sciences of the Organization for Drug ADR Relief, R&D Promotion and Product Review of Japan, and the Japan Health Sciences Foundation (Grants 341-5 and 321-2).

² Address correspondence and reprint requests to Dr. Mitsuo Honda, Vaccine Research and Development Group for Retroviruses, AIDS Research Center, National Institute of Infectious Diseases, Shinjuku-ku, Tokyo 162, Japan.

³ Abbreviations used in this paper: PND, principal neutralizing determinant; Thy/Liv SCID-hu, thymus/liver-transplanted severe combined immunodeficient mice; Rµ5.5, humanized antibody from anti-PND monoclonal antibody µ5.5; CDR, complementarity determining regions; FRs, framework regions; REI, region I; SGI, subgroup I; HCMV, human cytomegalovirus; TCID₅₀, 50% tissue culture infective dose; JCI, Japanese clinical isolate.

Received for publication May 5, 1997. Accepted for publication September 16, 1997.

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