Saliva inhibits HIV-1 infectivity

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Saliva inhibits HIV-1 infectivity P h ilip C. F o x , D D S A ndy W o lff, D M D C h ih -K o Y eh, BD S, P h D J a n e C. A tk in so n , D D S Bruce J. B au m , D M D , P h D

u m an im m unodeficiency virus 1 (HIV-1), the causative agent of the acqu ired im m u n e defi­ ciency syndrom e (AIDS), is infrequently rec o v ered fro m sa liv a ry s e c r e tio n s .1'2 D en ta l h e a lth care w o rk e rs also have been sh o w n to have an extrem ely low o c c u p a tio n a l risk o f a c q u ir in g HIV-1 infection.3 A dditionally, epidem iological data an d an intensive study of household contacts4,5 fail to su p p o rt a role for saliva in tran sm issio n of AIDS. T hese obser­ vations, however, have n o t quieted public o r p ro fessional an x iety co n c ern in g the risk of AIDS b ein g tra n sm itte d by the o ra l ro u te . A p r e lim in a r y r e p o r t has show n th at w hole saliva from ch im p an ­ zees an d h um ans could in h ib it infection o f h u m a n ly m p h o c y te s by H IV -1 .6 In the present study, the ability of h um an w hole saliva and individual gland secre­ tions from the p aro tid an d su b m an d ib ­ u la r/su b lin g u a l glands to in h ib it HIV1 in fe c tiv ity w as in v e s tig a te d . A fter in cu b atio n of HIV-1 w ith w hole or gland salivas for 1 h o u r, the ab ility of HIV1 to in fect ly m phocytes was decidedly reduced. T h is an tiv iral activity may be an im p o rtan t com ponent of oral defense against HIV-1 infection and may explain, in part, the lack of transm ission of AIDS by oral fluids.

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Methods and materials

Saliva sam ples were obtained from three h e a lth y , n o n m e d ic a te d , m a le , w h ite volunteers, aged 35, 40, an d 42, w ho do n o t b e lo n g to an y p o p u la tio n g r o u p know n to be at risk for AIDS. Samples w ere collected betw een 9:30 a n d 11:00 am . V o lu n te e rs h a d receiv ed n o th in g orally for 2 hours before saliva collection. U nstim ulated w hole saliva was collected f irs t by e x p e c to ra tio n in to a c h ille d c e n tr if u g e tu b e p la c e d o n ice. N ex t, p a ro tid an d s u b m a n d ib u la r/su b lin g u a l (hereafter referred to as subm andibular) salivas were collected separately, on ice, d u r in g 2% c itra te s tim u la tio n as p r e ­ viously described.7 T o each saliv a sa m p le (0.1 m L ), an e q u a l v o lu m e o f H IV -1 w as ad d e d as a 1:10 d ilu tio n in g ro w th m e d iu m of a p p ro x im a te ly 3 to 4 logioT C ID so/m L (TCIDso = tissue culture infectious dose yielding 50% infection). T h e m ixtures were incubated 1 h o u r at 37 C. A negative c o n tro l c o n s is tin g o n ly of g ro w th m edium (RPMI-1640 m edium co n tain in g 20% fetal calf serum, 100 U /m L p en icillin G , 100 jug /rn L s tre p to m y c in , 2 mM g lu ta m in e , 10% h u m a n in te rle u k in -2 , 2 /ug/m L polybrene, an d 20 U /m L a n ti­ alp h a interferon) an d a positive control

consisting of grow th m edium co n tain in g the sam e c o n c e n tra tio n of HIV-1 used w ith saliva sam ples, were in cu b ated in parallel. After in c u b a tio n , saliva sam ples an d the positive co n tro l were filtered th ro u g h 0.45 /urn nalgene filters, an d serial tenfold d ilu tio n s (u p to 10~4) w ere perform ed. T h e re a fte r, p h y to h e m a g g lu tin in - a c ti­ vated n o rm a l h u m a n p e rip h e ra l b lood lym phocytes ( ~ 6 x 106) were suspended in 1.0 m L of each d ilu tio n of the saliv a/ HIV-1 m ix tu re s, a n d the p o sitiv e an d negative control solutions, an d incubated fo r 1 h o u r a t 37 C to p e r m it v iru s ad so rp tio n . After this in c u b a tio n , each cell su sp e n sio n w as d ilu te d to 6.0 m L w ith grow th m edium , equally distributed in to th ree 16- x 25-m m c u ltu re tubes, an d further in cubated at 37 C in a 15% CC>2/95% a ir a tm o s p h e re o v e rn ig h t. S u p ern atan t flu id was carefully removed fro m ea c h c u ltu r e a n d fre sh g ro w th m edium (2.0 m L ) added to cells the next day (day 2), as w ell as o n days 8, 11, 15, an d 18. T h e release of infectious HIV1 from cells in to the su p e rn a tan t fluid w as fo llo w e d by m e a s u r in g reverse tra n s c r ip ta s e (R T ) a c tiv ity a t v ario u s tim e s, as in d ic a te d in T a b le 1. T h e m ethods used here for HIV-1 infection of target lym phocytes, an d m o n ito rin g JADA, Vol. 116, May 1988 ■ 635

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Table 1

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Inhibition of HIV-1 infectivity by saliva. R ev erse tra n s c rip ta s e

Experiment 1 S u b je c t 1§ W h o le s aliv a P a r o tid s a liv a S u b m a n d ib u la r s a liv a

I n h ib i tio n (%)*

H IV -1 ti te r + t (TC ID so)

a c tiv ity (cp m ± s e m ) t

D ay 8 100

D ay 18 70

0 100

0 54

P o sitiv e c o n tro l N e g a tiv e c o n tro l

D ay 8

D ay 18

D ay 8 861 ± 5 1 1 21,740 ± 4,867 1,774 ± 7 4 1

7,552 ± 3,793 18,429 ± 1,131

23,364 ± 2,543

18,814 ± 3 ,9 7 0

1,308 ± 3 0 7

182 ± 12

3,895 ± 3 ,7 1 7

0 10*83 0

D ay 18 10°.«J i0 J.»3 101-28

10'.5° 0

IO280 0

Experiment 2 S u b je c t 1§ W h o le s aliv a

D ay 15

D ay 18

100 0 100

100 13 100

58,560 ± 18,849 730 ± 58

100

100

0 54

0 14

1,007 ± 1 5 7 77,978 ± 18,226 1,692 ± 8 1 2

100 100

100 100 100

P a r o tid saliv a S u b m a n d ib u la r s aliv a S u b je c t 2 W h o le s aliv a P a r o tid saliv a S u b m a n d ib u la r s aliv a

D ay 15 843 ± 16

D ay 18 555 ± 55 27,108 ± 4 ,2 4 0 632 ± 94

466 ± 86 78,605 ± 19,845 8,502 ± 7,547

D ay 15 0 102 0

17

0 IO250 1 0 '.«

D ay 18 0 102 0

17

0 102'83 102 14

S u b je c t 3 W h o le s a liv a P a r o tid saliv a S u b m a n d i b u la r s a liv a

100

P o sitiv e c o n tro l N e g a tiv e c o n tro l

856 ± 153 1,088 ± 8 6 1,041 ± 6 0

1,015 ± 158

132,023 ± 20,446 804 ± 173

43,331 ± 5,082 892 ± 188

982 ± 33 592 ± 18

0 0 0 102 16 0

0 0 0 IO250 0

•Calculated from the TCID o virus titer on the day indicated. Results are the mean of triplicate determinations with each sample, as indicated in Methods and materials. Data represent the percentage inhibition of HiV-1 infectivity (measured by reverse transcriptase assay). The value of 100% indicates no HIV-1 growth (equal to the negative controls), and the value of 0% indicates HIV-1 growth equivalent to that observed with positive controls. -(•Results are the mean counts per m inute (± standard error) of the triplicate cultures at the first dilution (10 ') of virus-salivary mixture after filtration. f tT ite r is calculated from the triplicate culture of four dilutions (10~l to 10"4) of virus-saliva m ixture after filtration according to the method of Reed and Muench. §This is the same subject, tested on two separate occasions with two separate saliva collections.

by R T assays, have been described and ac c e p te d .8-11 T h e titers of HIV -1 were ca lc u lated a c c o rd in g to th e m eth o d of Reed and M uench.12

Results Prelim inary experim ents were conducted to d eterm in e w h e th e r in c u b a tio n w ith saliv a, u n d e r the c o n d itio n s described here w ould be toxic to h u m a n peripheral b lo o d ly m p h o c y te s. T h e v ia b ility an d g ro w th rates of lym phocytes incubated w ith saliva were w ith in 10% of control c e lls in c u b a te d w ith m e d iu m a lo n e , suggesting the absence of toxicity. T h e ab ility of saliva sam ples from a single s u b je c t ( e x p e rim e n t 1 in T a b le 1) to in h ib it the infectivity of HIV-1 was then e x a m in e d . At th e e a r lie s t tim e p o in t m easured (day 8), w hole saliva com pletely in h ib ited the ability of HIV-1 to infect h u m an lym phocytes. N o R T activity was o bserv ed in any o f th e c u ltu r e s u p e r­ n atan ts at this time. P arallel experim ents assessed the abilities of g la n d u lar secre­ tions (p arotid an d su b m an d ib u lar salivas) to in h ib it H IV -1 in fec tiv ity . S u b m a n ­ d ib u lar saliva was com parable to w hole saliva in its inhibitory activity, whereas n o in h i b i t i o n w as seen w ith p a r o tid saliva. L ym phocyte cu ltu re supernatants 636 a JADA, Vol. 116, May 1988

were also assayed for R T activity at day 18 a n d g e n e ra lly s im ila r fin d in g s , as those of day 8, were observed. A second e x p e rim e n t was co n d u cted in w hich sam ples from subject 1 (using a n o th e r set of fresh ly co llected saliva) were tested ag a in for HIV-1 in h ib ito ry activ ity a lo n g w ith sam p les from tw o other volunteers. For these experim ents, c u ltu re su p e rn a ta n ts from day 15 an d day 18 were assayed for R T activity (day 8 s a m p le s w ere n o t u sed b ec au se of technical problem s). S ubject 1 show ed the previously observed p attern of salivary in h ib itio n of HIV-1 infectivity. O n days 15 and 18, both w hole an d su b m an d ib u lar sa liv a sh o w ed c o m p le te in h ib itio n of HIV-1 infectivity. N o in h ibitory activity was detected w ith th is subject’s paro tid sa liv a sa m p le o n day 15, b u t a sm a ll degree of in h ib itio n (13%) was observed at day 18 w hen com pared w ith control viral grow th. W hole saliva sam ples from subjects 2 an d 3 w ere also c a p ab le of com pletely in h ib itin g HIV-1 infectivity (100% at both tim e points). T h e pattern o f g la n d sa liv a effects, h o w ev e r, w as d iffe re n t fo r th e se in d iv id u a ls . B oth subjects showed the presence of inhibitory activity in su b m an d ib u lar salivas (subject 2, p a r tia l; su b je c t 3, c o m p le te ). C o n ­ versely, a lth o u g h su b ject 2 show ed no

in h ib ito ry activity in paro tid saliva, the p a ro tid sam p le from subject 3 show ed com plete in h ib itio n of HIV-1 infectivity in b o th day 15 an d 18 assays. In further experim ents, w hen samples of w h o le sa liv a w ere filte re d b efo re in c u b a tio n w ith HIV-1 the in h ib ito ry activity was abolished com pletely (data n o t show n). S a m p lin g of su p e rn a tan ts from days 8, 11, 15, 18, 22, 25, and 29 rev e ale d n o r e d u c tio n in R T a c tiv ity com pared w ith control cultures.

Discussion T h e present study has dem onstrated that th e a b ility of HIV-1 to in fect h u m a n p e r ip h e ra l b lo o d ly m p h o c y te s can be c o m p letely in h ib ite d by in c u b a tio n of the virus in u n stim u la ted w hole saliva in vitro for 1 h o u r at 37 C. Im portantly, this finding, w hich is sim ilar to results p re lim in a rily described by F u ltz,6 was e x te n d e d to in d iv id u a l m a jo r g la n d secretions. In all subjects studied, in cu ­ bation of virus in subm andibular saliva resulted in a decided reduction of infec­ tivity. T h is fin d in g d em o n strate d th a t the in h ib ito ry activity fo u n d in w hole saliva is secreted by m ajor salivary glands an d does n o t exist in w hole saliva sim ply because of leakage of serum com ponents

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th rou gh oral le sio n s or th rou gh the gingival crevice. T h e presence o f in h ib ito ry activity in parotid saliva was variable; only one of three p arotid sam ples sh ow ed s ig ­ nificant inhibition of HIV-1. This may represent a general variation between individuals or specific differences unique to this individual. Further studies w ill clarify this p oin t. T h e fact that some parotid saliva samples showed no inhib­ itory activity toward HIV-1 was a sig ­ n ifica n t fin d in g . It argues that the inhibitory activity is likely not related to relatively nonspecific factors (salivary p H , io n ic strength), w h ich w ou ld be similar between gland secretions. Rather, it suggests that som e m acrom olecular com ponent is involved. T his suggestion is also supported by the observation that after filtration of w hole saliva through a 0.45-/um filter, in h ib ito ry activity toward HIV-1 was lost. It cannot be speculated as to the size of the an tiviral com p on en t from this ex p erim en t, how ever, because of the a b ility o f saliva to form large a ggregates and avid ly b in d sm aller com p onents. A dd itional studies, w ith larger numbers of subjects, are necessary to clarify the exact source (cell type), tim e k inetics, and chem ical nature of the salivary inhibitory activity against HIV-1. T here are m any p ossib le can ­ didates, including proteases and a number of salivary glycoproteins w hich possess antimicrobial activity. Alternatively, the a n tiviral com p on en t(s) may represent a previously undescribed factor. Exper­ im ents p resen tly are b ein g conducted to identify the antiviral component. Con­ ceivably, understanding the nature of the salivary an tiviral activity may be useful in developing therapeutic measures to control HIV-1 infection. T h e presence o f a factor in hum an

saliva that can inhibit HIV-1 infectivity is sig n ific a n t in m any respects. In particular, it likely provides a biologic ex p la n a tio n for the considerable, but indirect, data that su ggest a low risk for the oral tra n sm issio n of HIV-1. Unstim ulated saliva continually bathes the oral cavity and any HIV-1 virus present in the mouth would be exposed to a salivary inhibitory factor(s) for a considerable tim e. It is w orth n o tin g that saliva does n o t co n ta in broad spectrum a n tiv ira l a ctiv ity , that this HIV-1 inhibition is specific. For example, cytom eg a lo v iru s has been sh ow n to survive incubation in w hole saliva for 2 hours at 37 C ,13 w hereas in fectio u s Epstein-Barr virus can be readily cu l­ tured from saliva.14 Additionally, hepa­ titis B virus-containing whole saliva has been sh ow n to co n ta in and transm it hepatitis B in experimental transmission studies.15

Conclusion There is considerable work required to characterize salivary HIV-1 inhibitory activity. However, the results of this study demonstrate that this antiviral activity likely has a major role in the oral defense against AIDS. ------------------------- J l i O A

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Dr. Fox is senior investigator and head, Clinical Studies Unit; Dr. W olff is visiting fellow; Dr. Yeh is v isitin g associate; Dr. A tkinson is dental staff fellow; and Dr. Baum is chief, Clinical Investigations and P a tie n t Care B ranch and c lin ic a l director, N a tio n a l In stitu te of D ental R esearch, N a tion al Institutes of H ealth. Address requests for reprints to Dr. Fox at the Clinical Investigations and Patient Care Branch, N ational Institute of Dental Research, N ational Institutes of H ealth, B uild ing 10, Room IB-21, Bethesda, MD 20892.

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hom osexual men at risk for AIDS. Science 226:447449, 1984. 2. H o, D.D., and others. Infrequency o f isolation o f H T L V -III virus from saliva in AIDS. N E ngl J Med 313:1606, 1985. 3. Klein, R.S., and others. Low occupational risk of hum an im m unodeficiency virus infection am ong dental professionals. N Engl J Med 318:86-90, 1988. 4. Mann, J.M., and others. Prevalence of HTLVIII/L A V in h o u se h o ld contacts o f p atien ts w ith confirm ed AIDS and controls in Kinshasa, Zaire. JAMA 256(6):721-724, 1986. 5. Friedland, G .H ., and others. L ack of trans­ m ission of H T L V -III/L A V infection to household c o n ta cts of p a tien ts w ith A ID S or A ID S -related com plex w ith oral candidiasis. N Engl J Med 314:344349,1986. 6. Fultz, P .N . C om pon en ts of saliva inactivate h u m an im m u n o d eficien cy virus. L ancet ii: 1215, 1986. 7. Fox, P.C., and others. Xerostomia: evaluation o f a sym ptom w ith increasing significance. JADA 110(4):519-525, 1985. 8. Poiesz, B.J., and others. Detection and isolation of type C retrovirus particles from fresh and cultured lym p h ocytes of a p a tien t w ith cu tan eou s T cell lym phom a. Proc Natl Acad Sci USA 77:7415-7419, 1980. 9. P op ovic, M., and others. D etection, isolation and continuous production of cytopathic retroviruses (HTLV-III) from patients w ith AIDS and pre-AIDS. Science 224:497-500, 1984. 10. G allo, R .C ., and others. Frequent detection and is o la tio n o f cytop ath ic retroviruses (H T L V III) from patients with AIDS and at risk for AIDS. Science 224:500-503, 1984. 11. S a la h u d d in , S.Z., and oth ers. I so la tio n of infectious HTLV-III from AIDS and ARC patients and health y carriers: a study of risk factors and tissue sources. Proc N atl Acad Sci U SA 82:55305534, 1985. 12. Reed, L.J., and Muench, H. A sim ple method o f estim ating 50 percent endpoints. Am J Hygiene 27:493-497, 1938. 13. Schupfer, P.C.; M urph, J.R.; and Bale, J.F. Survival of cytom egalovirus in paper diapers and saliva. Pediatric Infect Dis 5:677-679, 1986. 14. M organ, D .G ., and others. Site o f E psteinBarr virus rep lication in the oropharynx. L ancet ii:l 154-1157, 1979. 15. A lter, H .J ., and oth ers. T r a n sm iss io n of hepatitis B to chim panzees by hepatitis B surface antigen p ositive saliva and sem en. Infect Im m un 16:928-933, 1977.

1. Groopman, J.E., and others. HTLV-III in saliva of people w ith AIDS-related com plex and healthy

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