N E W S A N D V I E W S

820 VOLUME 9 | NUMBER 7 | JULY 2003 NATURE MEDICINE

cross-reactive with PrPSc from mice, ham-sters, sheep, cattle and humans.

The Tyr-Tyr-Arg antibodies were not com-pletely TSE-specific because they could alsoimmunoprecipitate partially denatured, butstill protease-sensitive, PrP from acid-treatednormal brain tissue. These and other resultsshowed that the Tyr-Tyr-Arg antibodies canselectively pull down not only the partiallyprotease-resistant PrPSc, but also other formsof PrP that are conformationally perturbed.This is notable because some models of TSEdisease involve the accumulation of mis-folded, apparently pathological forms of PrPthat lack the full-blown protease-resistance oftypical PrPSc. These new antibodies maytherefore enable the recognition of a widerange of disease states associated with PrPmisfolding.

The antibodies seem to work well in teststhat can be transferred to the clinic. They rec-ognized PrPSc not only by immunoprecipita-tion, but also in a sandwich ELISAimmunoassay of scrapie brain homogenates.Moreover, brain tissue might not even beneeded for diagnostic purposes because theseantibodies can detect PrPSc in mouse spleenhomogenates by immunoprecipitation andin sheep lymph node follicular dendritic cellsby flow cytometry. Thus, peripheral tissues,which may be easier to biopsy than the brain,could serve as useful diagnostic specimens.

The full extent of the selectivity of the anti-bodies for PrPSc over PrPC and other proteinsremains to be determined. This is a practicalissue, because with many diagnostic orexperimental samples it will be desirable to

detect minute quantities of PrPSc among vastexcesses of potential competitors with Tyr-Tyr-Arg motifs. Nevertheless, the presentdata suggest that the new antibodies could bea boon to studies of the tissue distribution,cellular trafficking and pathological func-tions of PrPSc.

Because the Tyr-Tyr-Arg epitope isexposed in PrPSc and not in PrPC, the anti-bodies should also serve as revealing probesof the poorly understood conformationalchange that occurs with PrPSc formation. Atpresent, it is not clear which of the Tyr-Tyr-Arg epitopes become exposed in PrPSc, butfurther studies should readily provideanswers.

Paramithiotis et al. also suggest that theirantibodies might be useful in TSE therapeu-tics or immunoprophylaxis. These moleculesselect for PrPSc under physiological condi-tions and might be able to block its interac-tions with PrPC or other ligands necessary forPrPSc propagation and neurotoxicity.Supporting this possibility are studies show-ing that antibodies to PrPC or both PrPC andPrPSc can reduce infectivity in scrapie-infected microsomes3, block in vitro conver-sion of PrPC to PrPSc (ref. 4) and clearscrapie-infected cells of their infections5,6.Moreover, when administered passively7 orexpressed as chimeric transgenes8, such anti-bodies delay the onset of disease in infectedmice. The Tyr-Tyr-Arg antibodies may alsotag PrPSc deposits or infected cells for clear-ance and destruction by opsonization orcomplement-mediated mechanisms.

A major hurdle in therapeutic applications

will be the delivery of antibodies to the brain,where TSE pathology occurs. Another poten-tial problem is cross-reactivity of the antibod-ies to normal host proteins, which couldinduce autoimmune disease. However, previ-ous in vivo trials with antibodies to PrP havenot caused apparent autoimmune disease inmice7,8 unless PrPC was expressed at very highlevels8. Furthermore, the specificity of theTyr-Tyr-Arg antibodies for the abnormalform of PrP should lessen the likelihood ofproblems associated with autoimmunity toPrPC. Regardless of their future therapeutic orprophylactic value, these new antibodies havebeen sorely needed.

1. Paramithiotis, E. et al. A prion protein epitope selec-tive for the pathologically misfolded conformation.Nat. Med. 9, 893–899 (2003).

2. Korth, C. et al. Prion (PrPSc)-specific epitope definedby a monoclonal antibody. Nature 390, 74–77(1997).

3. Gabizon, R., McKinley, M.P., Groth, D. & Prusiner,S.B. Immunoaffinity purification and neutralization ofscrapie prion infectivity. Proc. Natl. Acad. Sci. USA85, 6617–6621 (1988).

4. Horiuchi, M., Chabry, J. & Caughey, B. Specific bind-ing of normal prion protein to the scrapie form via alocalized domain initiates its conversion to the pro-tease- resistant state. EMBO J. 18, 3193–3203(1999).

5. Enari, M., Flechsig, E. & Weissmann, C. Scrapie prionprotein accumulation by scrapie-infected neuroblas-toma cells abrogated by exposure to a prion proteinantibody. Proc. Natl. Acad. Sci. USA 98, 9295–9299(2001).

6. Peretz, D. et al. Antibodies inhibit prion propagationand clear cell cultures of prion infectivity. Nature412, 739–743 (2001).

7. White, A.R. et al. Monoclonal antibodies inhibit prionreplication and delay the development of prion dis-ease. Nature 422, 80–83 (2003).

8. Heppner, F.L. et al. Prevention of scrapie pathogene-sis by transgenic expression of anti-prion protein anti-bodies. Science 294, 178–182 (2001).

Dengue immune response: low affinity, high febrilityRaymond M Welsh & Alan L Rothman

Immunity built up after dengue virus infection protects only poorly against reinfection by a virus of a differentserotype, and second infections are often even more severe. A new study examines why (pages 921–927).

The immune response to viruses treads a fineline between damage to the host and controlof the pathogen. In the face of this delicatebalance, partial or incomplete immunity to apathogen can sometimes be worse than noimmunity. Nowhere in nature is this more

The authors are in the Departments of Pathology

and Medicine, University of Massachusetts Medical

School, Worcester, Massachusetts 01655, USA.

e-mail: Raymond.Welsh@umassmed.edu

apparent than in dengue virus infection.Dengue is a mosquito-borne tropical diseasecaused by any of four serotypes of the fla-vivirus of the same name. Immunity to agiven dengue virus serotype provides goodprotection against reinfection by that sameserotype. However, subsequent infection withother viral serotypes markedly increases therisk for dengue hemorrhagic fever (DHF) anddengue shock syndrome, the severe forms ofdengue virus infection marked by abnormalvascular permeability1.

In this issue, Mongkolsapaya et al. reportthat a substantial fraction of T cells activatedduring the second infection have poor affinityfor the antigenic peptides of this second virusserotype2. Instead, these T cells have higheraffinity to other (presumably previouslyencountered) dengue virus serotypes. Theauthors suggest that these ‘inappropriate’ Tcells contribute to immunopathology whiledoing little to clear the virus.

Dengue has not attracted widespreadattention from researchers in the developed

©20

03 N

atu

re P

ub

lish

ing

Gro

up

h

ttp

://w

ww

.nat

ure

.co

m/n

atu

rem

edic

ine

N E W S A N D V I E W S

NATURE MEDICINE VOLUME 9 | NUMBER 7 | JULY 2003 821

world, but it is a major public health concernin tropical areas. Worldwide, hundreds ofthousands of people contract DHF every year,and often require hospitalization to preventdeath; most of these cases occur during sec-ond dengue virus infections. The number ofcountries affected by dengue and the numberof cases of DHF have increased over the lastseveral decades3. There is an urgent need forvaccines against dengue virus, but the theo-retical potential for a vaccine to induce adetrimental immune response, enhancing therisk for DHF, has been a serious impedimentto its development.

These small flaviviruses encode only tenproteins, and the serotypes have high aminoacid sequence homology. Dengue virusserotypes can be distinguished by antibodyneutralization assays, but they elicit antibody

and T-cell responses that cross-react againstother serotypes. Classic studies by Halsteadand others led to the hypothesis that infectionwith a second dengue virus serotype results inantibody-mediated immune enhancement4.In this process, non-neutralizing antibody tothe first dengue serotype binds to the secondserotype.

This binding enhances infection of phago-cytic cells by the second serotype through anantibody (Fc) receptor entry route4.Antibody-mediated enhancement of infec-tion also can explain the occurrence of DHFduring primary dengue virus infection ininfants; this is typically observed in the sec-ond 6 months of life, when dengue-specificantibodies acquired from the mother declinebelow protective (neutralizing) levels.

The pathogenesis of DHF cannot entirely

be explained by antibody, however. DHF isassociated with high levels of T-cell cytokinessuch as IFN-γ and TNF-α, and evidence ofmarked T-cell activation in addition to highviremia levels5. To study T-cell responses aftersecond dengue virus infections, the investiga-tors first identified a peptide epitope recog-nized in the context of HLA-A*1101, an allelecommon in the Thai population. As with pre-viously described T-cell epitopes, the corre-sponding epitopes from the different denguevirus serotypes showed highly similar but notidentical sequences, indicating a potential forpartial cross-reactivity6.

In several patients, the authors foundthat the CD8+ T cells generated duringinfection bound weakly to major histocom-patibility complex tetramers presentingepitopes of the infecting virus, but boundmore strongly to other epitopes, presum-ably from previously encountered viruses.A high frequency of these T cells inducedduring the second infection displayed anapoptotic phenotype and seemed destinedto die before adequately controlling theinfection.

The authors interpreted this phenome-non as ‘original antigenic sin’ of T cells,whereby cross-reactive T cells to onepathogen dominate a response to a relatedpathogen7,8. Cross-reactive T cellsexpanded by the previously encountereddengue virus serotype may prevail over Tcells with higher affinity for the newserotype because of their numerical advan-tage and memory phenotype (Fig. 1). Thisphenomenon of immunodomination mayoccur by T-cell competition for antigen onthe antigen-presenting cells9, and is animportant aspect of the heterologous T-cellimmunity that can result from cross-reac-tive T-cell responses, even between rela-tively unrelated viruses10.

An alternative explanation for theseobservations is that enhanced viral replica-tion caused by antibody-mediated immuneenhancement may drive the T cells withhighest affinity for the infecting virus intoapoptosis, through the process of activa-tion-induced cell death11 (Fig. 1).Definitive evidence of one or both of thesemechanisms is likely to come from prospec-tive cohort studies. By either mechanism,the host is left with a weak and ineffectivegroup of T cells to clear the infection.

If these detrimental T cells contribute toDHF pathogenesis, might their role involvemechanisms other than inefficient viralclearance? This seems likely, because animalmodels have shown that immunopathologyoccurs when T cells are of low affinity and

D2

Rel

ativ

e af

fin

ity

D3 Dengue 2 Dengue 3

Dengue 2 → 3

Expansion due tocross-reactivity

Inhibition due to AICDor immunodomination

Inefficient viralclearance

Enhancedimmunopathology(DHF)

Primary infection

Sequential infectionD2

Rel

ativ

e af

fin

ity

D3

a

b

Figure 1 Low-affinity T-cell response to dengue antigens after secondary (sequential) infection. Circlesrepresent the distribution of T-cell clones of different affinities to cross-reactive peptide epitopes. (a)Serotype-specific responses tend to select for higher affinity T cells during acute primary infections. (b)Upon infection with a second virus serotype, the response is dominated by T cells of higher affinity tothe first encountered virus (in this example, dengue 2). This could be due to a preferential expansion ofpre-existing low-affinity memory T cells. Alternatively, the higher affinity cells may be driven intoactivation-induced cell death (AICD) by the high antigen load caused by antibody-mediatedenhancement of infection. Whatever the mechanism, the consequences may be inefficient viralclearance and enhanced immunopathology in the form of DHF. D2, dengue 2; D3, dengue 3.

M.A

. Bre

hm

©20

03 N

atu

re P

ub

lish

ing

Gro

up

h

ttp

://w

ww

.nat

ure

.co

m/n

atu

rem

edic

ine

N E W S A N D V I E W S

822 VOLUME 9 | NUMBER 7 | JULY 2003 NATURE MEDICINE

continue to secrete cytokines in the presenceof residual antigen12. Mouse models of het-erologous immunity have similarly shownmarked changes in pathology when T cellsspecific to previously encountered virusesalter the response to subsequent virus infec-tions10.

Understanding the relationship betweenpositive and negative effects of the T-cellresponse to dengue virus infections mightpermit the design of vaccines to minimize theinduction of low avidity cross-reactiveimmune responses. The need for a safe andeffective vaccine is certainly great, and it is tobe hoped that the application of newimmunologic techniques, such as those used

by the authors, will provide the necessaryclues.

1. Thein, S. et al. Risk factors in dengue shock syndrome.Am. J. Trop. Med. Hyg. 56, 566–572 (1997).

2. Mongkolsapaya, J. et al. Original antigenic sin andapoptosis in the pathogenesis of dengue hemorrhagicfever. Nat. Med. 9, 921–927 (2003).

3. Gubler, D.J. Dengue and dengue hemorrhagic fever.Clin. Microbiol. Rev. 11, 480–496 (1998).

4. Morens, D.M. Antibody-dependent enhancement ofinfection and the pathogenesis of viral disease. Clin.Infect. Dis. 19, 500–512 (1994).

5. Libraty, D.H. et al. Differing influences of viral burdenand immune activation on disease severity in secondarydengue 3 virus infections. J. Infect. Dis. 185,1213–1221 (2002).

6. Zivny, J. et al. Partial agonist effect influences the CTLresponse to a heterologous dengue virus serotype. J.Immunol. 163, 2754–2760 (1999).

7. Klenerman, P. & Zinkernagel, R.M. Original antigen sin

impairs cytotoxic T lymphocyte responses to virusesbearing variant epitopes. Nature 394, 482–485(1998).

8. Brehm, M.A. et al. T cell immunodominance and main-tenance of memory regulated by unexpectedly cross-reactive pathogens. Nat. Immunol. 3, 627–634(2002).

9. Yewdell, J.W. & Bennink, J.R. Immunodominance inmajor histocompatibility complex class I-restricted Tlymphocyte responses. Annu. Rev. Immunol. 17,51–88 (1999).

10. Welsh, R.M. & Selin, L.K. No one is naive: the signifi-cance of heterologous T-cell immunity. Nat. Rev.Immunol. 2, 417–426 (2002).

11. Combadiere, B., Sousa, C.R., Germain, R.N. &Lenardo, M.J. Selective induction of apoptosis inmature T lymphocytes by variant T cell receptor ligands.J. Exp. Med. 187, 349–355 (1998).

12. von Herrath, M.G. & Oldstone, M.B. Interferon-gammais essential for destruction of beta cells and develop-ment of insulin-dependent diabetes mellitus. J. Exp.Med. 185, 531–539 (1997).

A recent report in Cancer Cell calls intoquestion the simple, elegant idea of stop-ping tumors with angiogenesis inhibitors.Pennaccietti et al.1 find that hypoxia,instead of inhibiting tumor growth, stimu-lates tumor invasion by activating hepato-cyte growth factor (HGF). The studysuggests that optimal antiangiogenic ther-apy may require complex solutions.

HGF, also known as scatter factor, bindsto the c-met proto-oncogene tyrosinekinase receptor. HGF signals invasive pat-terning in normal tissues such as axons andthe mammary gland. In cancer, overexpres-sion and mutation of HGF pathway mem-bers have been implicated in the origin orprogression of many cancer types, includ-ing renal, breast, head and neck and col-orectal carcinomas and melanoma2.

Reasoning that angiogenesis and invasionpathways work hand in hand to producelethal metastases, Pennacchietti et al. exam-ined the possible interplay between hypoxiaand HGF-induced invasion. In normal and

tumor cell lines, the authors found thathypoxia elevated HGF mRNA and proteinexpression. In agreement with this finding,maximal HGF protein expression in tumorsections, as determined by immunofluores-cence, occurred in areas distant frommicrovessels. These areas were also thoughtto be oxygen-poor based on high hypoxia-inducible factor-1α staining.

The investigators next examined thefunctional consequences of elevated HGFexpression. They found that HGF andhypoxia activated c-met synergistically.Downstream of c-met activation, HGF andhypoxia elevated motility and invasion inthree in vitro assays: cell scattering, invasionof Matrigel-coated filters and branchingmorphogenesis in collagen. The branchingassay was conducted in the presence ofserum, showing that HGF can exert a domi-nant effect in the milieu of factors thattumor cells encounter. Moreover, RNAinterference inhibition of c-met abrogatedthe functional effects of hypoxia and HGF.The authors concluded that hypoxia canstimulate HGF-driven tumor cell invasion.

The current study did not test whetherangiogenesis inhibitors stimulate HGF-driven tumor cell invasion in the same waythat hypoxia does. Nevertheless, the authorsdiscuss the possiblity that induction of

hypoxia by antiangiogenic strategies mayhave unintended consequences such as thestimulation of invasion. They propose thatan “invasive switch” in response to hypoxiapermits tumor cells to escape and survive.

This notion is consistent with variousother studies that have linked hypoxia totumor aggressiveness through several inter-mediaries: increased production ofautocrine motility factor and increasedexpression of tumor urokinase plasmino-gen activator receptor3, and a proteasereceptor. Moreover, a correlation of hypoxicstatus in vivo with increased metastaticspread has been observed in KHT-Cfibrosarcoma cells and D-12 melanomacells5,6.

This report comes on the heels of a man-uscript by Yu et al. in Science, documentingcomplex outcomes of antiangiogenic ther-apy7. These investigators tested the effectsof tumor cell p53 status, which is thought toinfluence apoptotic decisions in response tohypoxia. They asked whether tumor cellp53 status influenced the outcome ofantiangiogenic therapy. They injectedeither wild-type or p53-null HCT116 col-orectal carcinoma cells into immunocom-promised mice, then treated them with acombination of low-dose chemotherapyand an antibody to vascular endothelial

Angiogenesis inhibitors: motivators of metastasis?Patricia S Steeg

Angiogenesis inhibitors have shown promise in hindering blood supply and holding tumors in check. But it nowseems that such inhibitors, by depriving tumors of oxygen, could have an unintended effect: promotion ofmetastasis.

The author is in the Women’s Cancers Section,

Laboratory of Pathology, Center for Cancer

Research, National Cancer Institute,

Bethesda, Maryland 20892, USA.

e-mail: steegp@mail.nih.gov

©20

03 N

atu

re P

ub

lish

ing

Gro

up

h

ttp

://w

ww

.nat

ure

.co

m/n

atu

rem

edic

ine