11. J. Arrington, I. Sick, J. Phys. Chem. Ref. Data 44, 031204
12. H world data corresponds to adjustment 8, table XXIX in ( 3).
13. R. Pohl et al., Science 353, 669–673 (2016).
14. B. de Beauvoir et al., Eur. Phys. J. D 12, 61–93 (2000).
15. B. de Beauvoir et al., Eur. Phys. J. D 14, 398 (2001).
16. D. J. Berkeland, E. A. Hinds, M. G. Boshier, Phys. Rev. Lett. 75,
17. Materials and methods are available as supplementary
18. M. Horbatsch, E. A. Hessels, Phys. Rev. A 82, 052519 (2010).
19. D. J. Berkeland, thesis, Yale University (1995).
20. A. Marsman, M. Horbatsch, E. A. Hessels, J. Phys. Chem. Ref.
Data 44, 031207 (2015).
21. G.-P. Feng, X. Zheng, Y. R. Sun, S.-M. Hu, Phys. Rev. A 91,
22. M. Horbatsch, E. A. Hessels, Phys. Rev. A 84, 032508 (2011).
23. A. Marsman, M. Horbatsch, E. A. Hessels, Phys. Rev. A 86,
24. A. Marsman, M. Horbatsch, E. A. Hessels, Phys. Rev. A 86,
25. A. Marsman, E. A. Hessels, M. Horbatsch, Phys. Rev. A 89,
26. D. C. Yost et al., Phys. Rev. A 90, 012512 (2014).
27. R. Loudon, The Quantum Theory of Light (Oxford Univ. Press,
28. U. D. Jentschura, P. J. Mohr, Can. J. Phys. 80, 633–644 (2002).
29. R. C. Brown et al., Phys. Rev. A 87, 032504 (2013).
30. U. Fano, Phys. Rev. 124, 1866–1878 (1961).
31. S. Schippers, Int. Rev. At. Mol. Phys. 2, 151–156 (2011).
32. S. Schippers, Analytical expression for the convolution of a
Fano line profile with a Gaussian. arXiv:1203.4281v2 [physics.
atom-ph] ( 13 May 2016).
33. A. Beyer et al., J. Phys. Conf. Ser. 467, 012003 (2013).
34. A. Beyer et al., Phys. Scr. T165, 014030 (2015).
35. A. Beyer et al., Ann. Phys. 525, 671–679 (2013).
36. A. Beyer et al., Opt. Express 24, 17470–17485 (2016).
37. C. J. Sansonetti et al., Phys. Rev. Lett. 107, 023001 (2011).
38. M. Horbatsch, E. A. Hessels, Phys. Rev. A 93, 022513
39. R. Pohl et al., Metrologia 54, L1–L10 (2017).
40. A. C. Vutha et al., Bull. Am. Phys. Soc. 57, D1.138 (2012)
41. S. Galtier et al., J. Phys. Chem. Ref. Data 44, 031201 (2015).
42. D. C. Yost et al., Phys. Rev. A 93, 042509 (2016).
43. M. Puchalski, J. Komasa, P. Czachorowski, K. Pachucki, Phys.
Rev. Lett. 117, 263002 (2016).
44. J. Liu et al., J. Chem. Phys. 130, 174306 (2009).
45. D. Sprecher, J. Liu, C. Jungen, W. Ubachs, F. Merkt, J. Chem.
Phys. 133, 111102 (2010).
46. J.-P. Karr, L. Hilico, J. C. J. Koelemeij, V. I. Korobov, Phys. Rev.
A 94, 050501 (2016).
47. R. K. Altmann, S. Galtier, L. S. Dreissen, K. S. E. Eikema, Phys.
Rev. Lett. 117, 173201 (2016).
48. U. D. Jentschura, P. J. Mohr, J. N. Tan, J. Phys. B 43, 074002
The authors thank E. A. Hessels and U. D. Jentschura for insightful
discussions and W. Simon, K. Linner, and H. Brückner for technical
support. K.K. and N.K. acknowledge support from Russian Science
Foundation 16-12-00096, R.P. from the European Research Council
(ERC) Starting Grant #279765, and T. W.H. from the Max Planck
Foundation. The data underlying this study are available from the
corresponding author upon reasonable request.
Materials and Methods
Tables S1 to S3
References ( 49–59)
28 July 2016; accepted 28 August 2017
Trispecific broadly neutralizing HIV
antibodies mediate potent SHIV
protection in macaques
Ling Xu,1 Amarendra Pegu,2 Ercole Rao,1 Nicole Doria-Rose,2 Jochen Beninga,1
Krisha McKee,2 Dana M. Lord,1 Ronnie R. Wei,1 Gejing Deng,1 Mark Louder,2
Stephen D. Schmidt,2 Zachary Mankoff,2 Lan Wu,1 Mangaiarkarasi Asokan,2
Christian Beil,1 Christian Lange,1 Wulf Dirk Leuschner,1 Jochen Kruip,1 Rebecca Sendak,1
Young Do Kwon,2 Tongqing Zhou,2 Xuejun Chen,2 Robert T. Bailer,2 Keyun Wang,2
Misook Choe,2 Lawrence J. Tartaglia, 3, 4 Dan H. Barouch, 3, 4 Sijy O’Dell,2 John-Paul Todd,2
Dennis R. Burton, 4, 5 Mario Roederer,2 Mark Connors, 6 Richard A. Koup,2 Peter D. Kwong,2
Zhi-yong Yang,1 John R. Mascola,2† Gary J. Nabel1†
The development of an effective AIDS vaccine has been challenging because of viral
genetic diversity and the difficulty of generating broadly neutralizing antibodies (bnAbs).
We engineered trispecific antibodies (Abs) that allow a single molecule to interact with
three independent HIV-1 envelope determinants: the CD4 binding site, the membrane-proximal external region (MPER), and the V1V2 glycan site. Trispecific Abs exhibited higher
potency and breadth than any previously described single bnAb, showed pharmacokinetics
similar to those of human bnAbs, and conferred complete immunity against a mixture of
simian-human immunodeficiency viruses (SHIVs) in nonhuman primates, in contrast to
single bnAbs. Trispecific Abs thus constitute a platform to engage multiple therapeutic
targets through a single protein, and they may be applicable for treatment of diverse
diseases, including infections, cancer, and autoimmunity.
Avariety of broadly neutralizing antibodies (bnAbs) have been isolated from HIV-1– infected individuals (1– 3), but their poten- tial to treat or prevent infection in humans may be limited by the potency or breadth
of viruses neutralized ( 4, 5). The targets of these
antibodies have been defined according to an
understanding of the HIV-1 envelope structure
( 6–9). Although bnAbs occur in selected HIV-1–
infected individuals (usually after several years
of infection), it remains a challenge to elicit them
by vaccination because broad and potent HIV-
1 neutralization often requires unusual antibody
characteristics, such as long hypervariable loops,
interaction with glycans, and a substantial level
of somatic mutation. Strategies have thus shifted
from active to passive immunization, both to protect against infection and to target latent virus
We and others have begun to explore com-
binations of bnAbs that optimize potency and
breadth of protection, thus reducing the likeli-
hood of resistance and viral escape ( 15–17). Anti-
bodies directed to the CD4 binding site (CD4bs),
membrane-proximal external region (MPER),
and variable-region glycans are among the com-
binations that so far provide optimal neutraliza-
tion ( 18). In addition, alternative combinations
have also been investigated for the immunotherapy
of AIDS, specifically by directing T lymphocytes
to activate latent viral gene expression and enhance
lysis of virally infected cells ( 19, 20). Because mul-
tiple antibodies may help to reduce the viral repli-
cation that sustains chronic HIV-1 infection, we
report here the generation of multispecific anti-
bodies designed to increase the potential efficacy
of HIV-1 antibodies for prevention or therapy.
Design of bispecific antibodies and
evaluation of neutralization breadth
Although individual anti–HIV-1 bnAbs can neutralize naturally occurring viral isolates with high
potency, the percentage of strains inhibited by
these monoclonal Abs (mAbs) varies ( 21, 22). In
addition, resistant viruses can be found in the
same patients from whom bnAbs were isolated,
which suggests that immune pressure against a
single epitope may not optimally treat HIV-1
infection or protect against it. We hypothesized
that the breadth and potency of HIV-1 neutralization by a single antibody could be increased
by combining the specificities against different
epitopes into a single molecule. This strategy
would be expected not only to improve efficacy
but also to simplify treatment regimens, as well
as the regulatory issues required for clinical
To test this concept, we initially incorporated
prototype bnAbs to the CD4bs and MPER sites
into a modified bispecific Ab. When two variable regions are linked in tandem, the distal
site typically retains its ability to bind antigen
while the proximal binding is markedly diminished. We therefore used an alternative configuration, termed CODV-Ig, that introduced linkers
and inverted the order of the antibody binding site in light and heavy chains to alter the