The spectrum of hematologic and immunologic abnormalities inhuman immunodeficiency virus type 1 (HIV-1) infection is broad1,2.Although the incidence of B-cell-associated neoplasms is increased,relatively few cases of multiple myeloma have been described3,4.There are, however, reports of patients in whom serum paraproteinspresent as single or oligoclonal bands5,6,7,8,9,10,11. Theseparaproteins have previously been thought of as part of theexuberant polyclonal response to HIV-110,11.
In this report, we describe an HIV-1-positive patient with myelomawhose IgG- paraprotein specifically recognized the HIV-1 p24gag antigen. It is possible that an HIV-1 antigen-driven responseplayed an important part in the pathogenesis of this patient'smyeloma.
Case Report
A 30-year-old bisexual man presented with acute renal failure,a blood urea nitrogen concentration of 72 mg per deciliter (26mmol per liter), and a creatinine concentration of 4.7 mg perdeciliter (415 µmol per liter). The uric acid concentrationwas 12.8 mg per deciliter (0.77 mmol per liter). His hemoglobinlevel was 10.3 g per deciliter, and the hematocrit 31 percent.The formation of rouleaux was noted.
The patient's serum osmolality was 312 mOsm per kilogram, hisserum total protein 12.8 g per deciliter, and his serum viscosity3.1 times that of water (normal, 1.4 to 1.8). Agarose-gel proteinelectrophoresis and immunofixation of the patient's serum demonstratedan IgG- paraprotein at a concentration of 6.4 g per deciliter.Urine protein electrophoresis and immunofixation demonstratedthe presence of 0.6 g of IgG- and free light chains per totalvolume.
A bone marrow biopsy revealed marked plasmacytosis, with manyplasmablasts present. The aspirate contained 34 percent plasmacells, including many immature and binucleate forms. On thebasis of the findings of serum and urine paraproteins, bonemarrow plasmacytosis, anemia, increased uric acid, and renalfailure, a diagnosis of multiple myeloma was considered.
Results
A test for HIV-1 antibody by enzyme immunoassay (Genetic Systems)was strongly positive. Western blot analysis (Bio-Rad) demonstratedmultiple reactive bands corresponding to different pol, env,and gag antigens. Western blotting of serial dilutions of thepatient's serum showed reactivity with p24 and p55 up to a dilutionof 1:10,000, with the reactivity stronger for p24 than for p55.
These results suggested that the serum paraprotein might bedirected against the HIV-1 p24 gag antigen. Since affinity chromatographyhas been used to characterize myeloma paraproteins,12 we testedthis hypothesis by passing the patient's serum over a p24 affinitycolumn constructed by coupling recombinant HIV-1 p24 antigen(a gift from Dr. C. DeBouck) to cyanogen bromide-activated Sepharose4B beads (Pharmacia).
The column was washed with 0.01 M phosphate (pH 7.4) in 0.15M sodium chloride (phosphate-buffered saline) until the effluenthad an optical density at 280 nm comparable to that of phosphate-bufferedsaline. The effluent was collected and concentrated. Bound antibodywas eluted with 4 ml of a glycine buffer (0.1 M, pH 2.0). Thefraction containing the eluted antibody was collected, neutralizedin TRIS (1.0 M, pH 8.0, 50 microl per milliliter of solution),and concentrated (1 mg per milliliter in phosphate-bufferedsaline).
Figure 1 shows densitometric scans of electrophoretograms ofthe patient's serum, the column effluent, and the eluted antibody.The serum paraprotein bound measurably to the p24 affinity column,as evidenced by a decrease in the concentration of paraproteinin the column effluent. When it was eluted from the column,the immunoadsorbed antibody was present as a single band. Immunofixationdemonstrated the affinity-purified antibody to be a single IgG-species that comigrated with the serum paraprotein (Figure 2).
Figure 1. Densitometric Scanning of Agarose-Gel Electrophoretograms of the Patient's Serum, the Affinity-Column Effluent, and Affinity-Purified Antibody.
Agarose-gel electrophoresis was performed on the patient's serum before (Panel A) and after (Panel B) passage through the recombinant p24 affinity column; it was also performed on 2 µg of eluted antibody (Panel C). The peaks at left correspond to albumin, and those at right to the paraprotein. A single passage through the affinity column selectively decreased the concentration of paraprotein from 39.2 percent to 30.0 percent of total serum protein.
Figure 2. Agarose-Gel Electrophoresis of the Patient's Serum and the Affinity-Purified Paraprotein Eluted from the Recombinant p24 Affinity Column.
Lane 1 shows the patient's serum; lane 2, 2 µg of the eluted antibody after migration with the serum paraprotein; and lanes 3 through 7, the results after the immunofixation of 2 µg of the eluted antibody for IgG, IgA, IgM, light chain, and light chain, respectively. This analysis demonstrated that the paraprotein was of the IgG- species. "Origin" denotes the point of application of the sample.
HIV-1 Western blot analysis of the eluted antibody to determineits specificity showed strong and weak reactivity, respectively,against the gag antigens p24 and p55. The fainter reactivityfor p55 was probably due to cross-reactivity between relatedgag gene products. Figure 3 shows that this pattern correspondedto those observed when the serum was serially diluted. In addition,the antibody was tested for reactivity with selected infectiousagents other than HIV-1; it did not react with cytomegalovirus,streptolysin O, Treponema pallidum, or Toxoplasma gondii. Together,these findings indicated that the paraprotein in this patientwas directed against the p24 antigen.
Figure 3. Western Blot Analysis of the Patient's Serum and the Eluted Antibody.
The serum samples are shown undiluted (lane 1) and at dilutions of 1:10 (lane 2), 1:100 (lane 3), 1:1000 (lane 4), 1:10,000 (lane 5), and 1:100,000 (lane 6). At the higher concentrations, the serum was reactive with many different gag, pol, and env antigens. At a dilution of 1:10,000 (lane 5), it remained reactive with gag antigens p24 and p55, reacting more strongly with p24. The serum was nonreactive at a dilution of 1:100,000 (lane 6). Western blot analysis of 2 µg and 0.2 µg of the eluted antibody is shown in lanes 7 and 8, respectively, and in both cases there was reactivity to the gag antigens p24 and p55, with a stronger reaction to p24. There was no reactivity with 0.02 µg of the eluted antibody (lane 9).
To investigate further the clonal nature of the immune response,Southern blot analysis was performed on cells aspirated fromthe bone marrow as described elsewhere13. This analysis clearlydemonstrated a clonal pattern of immunoglobulin gene rearrangement(Figure 4).
Figure 4. Southern Blot Analysis of the Patient's Bone Marrow Aspirate.
Samples of control tonsil DNA (lanes 1 and 3) and the bone marrow aspirate (lanes 2 and 4) were doubly digested with the restriction endonucleases EcoRI and BamHI, each of which was paired with HindIII. The digests were then hybridized with a 3.3-kb JH fragment radiolabeled with phosphorus-32. Analysis of the aspirate with EcoRI and HindIII demonstrated two rearranged bands (R) in addition to the germline band (G) present in tonsil DNA. After digestion with BamHI and HindIII, a rearranged band was also present in addition to the germline band.
In addition, cytospin preparations of the aspirate that hadbeen fixed in methanol at 4 °C for 30 minutes were studiedby fluorescent immunochemistry. The vast majority of the plasmacells contained IgG- (more than 90 percent of those stainedwith anti- as compared with less than 5 percent of those stainedwith anti-micro; more than 90 percent of those stained withanti- as compared with less than 5 percent of those stainedwith anti-). Flow-cytometric analysis of a sample of permeabilizedaspirated cells confirmed these results by demonstrating anoverall :micro ratio of 8:1 and a : ratio of 7:1.
Discussion
It appears that the paraprotein of this HIV-1-infected patientwith myeloma was directed specifically against the HIV-1 p24antigen. The evidence of specificity stems from the measurableand selective immunoadsorption of the paraprotein from the patient'sserum by a recombinant p24 affinity column. The affinity-purifiedantibody consisted of a single IgG- species that comigratedwith the serum paraprotein.
Western blot analysis demonstrated antibody reactivity againstHIV-1 gag antigens p24 and p55. Southern blot analysis, immunochemicalanalysis, and flow-cytometric analysis performed on a bone marrowaspirate strongly suggested a monoclonal process, although thepossibility that more than one clone existed cannot be excludedwithout immunoglobulin sequencing.
It is unusual to identify a paraprotein in an HIV-1-positivepatient with myeloma that is directed against one specific HIV-1antigen. There have been previous reports of myeloma paraproteinsdirected against a wide variety of infectious agents, includingbacteria such as streptococcus14,15,16,17. These studies suggestthe existence of a causal relation between myeloma and antigenicstimulation, reminiscent of an earlier hypothesis that B-cellcancers may also be related to antigenic stimulation18,19.
Free p24 antigen is usually present in the circulation shortlyafter infection with HIV-1 and before the development of a humoralresponse20,21. We suggest that the lymphoproliferative diseasedeveloped in this patient at least partly because of an antigen-drivenresponse to circulating p24 antigen. The specificity of thismyeloma-associated paraprotein for p24 suggests that clonalselection by HIV-1 antigen played a part in the pathogenesisof this plasmacytic disorder.
We are indebted to Ms. Kathy Bayer, Ms. Mattie Gadson, and Ms.Emily Thompson for their expert assistance; to Drs. David Biggs,Marc Kahn, and Robert Rotche for obtaining bone marrow aspirates;and to Drs. Marilyn Senior, Bonnie Mochan, and Lorna Lynn fortheir assistance.
Source Information
From the Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, 6 Founders Bldg., 3400 Spruce St., Philadelphia, PA 19104-6082, where reprint requests should be addressed to Dr. Silberstein.
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paraprotein specifically recognized the HIV-1 p24 gag antigen. It is possible that an HIV-1 antigen-driven response played an important part in the pathogenesis of this patient's myeloma. 
as compared with less than 5 percent of those stained with anti-micro; more than 90 percent of those stained with anti-
). Flow-cytometric analysis of a sample of permeabilized aspirated cells confirmed these results by demonstrating an overall 
-staphylolysin in serum samples from a patient with multiple myeloma. Acta Pathol Microbiol Scand 1965;65:303-310. [Medline]
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