Several groups have recently developed hmAbs to the SARS CoV S glycoprotein that neutralize the virus and have potential for therapy and prophylaxis of SARS (reviewed in [84
]) .Recently, an improved method for Epstein-Barr Virus (EBV) transformation of human B cells has been developed based on CpG oligonucleotide (CpG 2006) that increases the B cell immortalization efficiency from 1–2% to 30–100%, and this method was used for selection of hmAbs specific for SARS CoV proteins [85
]. One of the selected antibodies (S3.1), which was specific for the S glycoprotein on the viral spikes, was about 500-fold more efficient in neutralization than convalescent serum. S3.1 prevented the cytopathic effect of the SARS CoV at 300 ng/ml [85
], and inhibited entry of pseudovirus with S glycoprotein from the Urbani isolate with about the same IC50
. However, it did not affect to any significant extent pseudovirus entry mediated by the GD03T0013 isolate S glycoprotein and even enhanced the entry of virus pseudotyped with the S glycoprotein from the palm civet isolate SZ16 [86
]. In a mouse model of SARS CoV infection this antibody prevented viral replication in the lower respiratory tract (at doses of 200 µg and 800 µg), and reduced it in the upper respiratory tract at the highest dose (800 µg) used. But, data for the in vivo neutralizing activity of other nhmAbs selected in this study [85
] including the most potent antibody (S215.13), which has a neutralizing concentration (1 ng/ml) 300-fold lower than that of S3.1, have not been reported in this article. The high neutralizing activities of these two hmAbs in IgG1 format indicates possibilities for their use alone or in combination for prophylaxis and treatment of SARS.
Phage display technology has been increasingly used to produce high affinity hmAbs from both naïve and immune libraries. An advantage of using a naïve library is that B lymphocytes from an infected or immunized host are not required. Recently, two human nonimmune scFv libraries containing about 1010
members were developed from B cells of unimmunized donors, and used for selection of antibodies against a purified S fragment containing residues 12–672 [87
]. One of the selected antibodies, IgG1 80R, can neutralize 50% of the virus in a microneutralization assay at a concentration as low as 0.37 nM. It also blocked formation of syncytia, which could contribute to the spread of the virus in vivo, although at significantly higher concentration (25 nM). Its epitope overlaps the binding site of the SARS CoV receptor ACE2 suggesting a possible mechanism of neutralization by preventing the virus attachment to its receptor [87
]. When 80R IgG1 was given prophylactically to mice at doses therapeutically achievable in humans, viral replication was reduced to below assay limits [89
]. One should note that the conditions used for evaluation of the neutralizing activity of different antibodies in this study are not exactly the same as in the study described above and also below; thus comparing the activity of different antibodies should be done with caution unless they are tested side by side under exactly the same conditions.
Three neutralizing hmAbs were also selected from another large naïve antibody library [90
]. They bound a recombinant S1 fragment comprising amino acid residues 318 to 510, a region previously identified as the SARS RBD (S RBD) [91
]. The most potent of these hnmAb, IgG1 CR3014, required the residue Asn479 of S RBD for its binding [91
]. This antibody exhibited in vitro 50% neutralizing activity at about 1 µg/ml. More importantly, this antibody showed neutralizing activity in ferrets. In one set of experiments ferrets were inoculated either with virus at two doses (low – 103
and high - 104
) or with virus preincubated with the antibody at 0.13 mg/ml for the low dose and 1.3 mg/ml for the high one. Animals exposed to the virus-antibody mixture had almost undetectable SARS CoV in the lung, showed no lung lesions on day 4 or 7, and did not shed virus in their throats unlike control animals treated with irrelevant antibody. In a second set of experiments the antibody at 10 mg/kg was administered 24 h before challenge with virus and reached 65–84 µg/ml serum concentration in three of the animals (< 5 µg/ml in the fourth one). In the three ferrets with high antibody concentration virus shedding in the throat was completely abolished while in the fourth one it was comparable to that of the control group. The CR3014 treated animals had 3.3 logs lower mean virus titer than the controls, and were completely protected from macroscopic lung pathology. Note that the antibody dose used (10 mg/kg) was less than the one (15 mg/kg) used for prevention of RSV infections in infants which is administered once a month. Recently, in vitro SARS-CoV variants escaping neutralization by CR3014 were generated that all had a single Pro462Leu mutation in the S glycoprotein of the escape virus [92
]. In vitro experiments confirmed that binding of CR3014 to a recombinant S fragment (amino acid residues 318–510) harboring this mutation was abolished. An antibody-phage library derived from blood of a convalescent SARS patient was screened for antibodies complementary to CR3014. A novel mAb, CR3022, was identified that neutralized CR3014 escape viruses, did not compete with CR3014 for binding to recombinant S1 fragments, and bound to S1 fragments derived from the civet cat SARS-CoV-like strain SZ3. No escape variants could be generated with CR3022. The mixture of both mAbs showed neutralization of SARS-CoV in a synergistic fashion by recognizing different epitopes on the RBD but the mechanism remains unknown. Dose reduction indices of 4.5 and 20.5 were observed for CR3014 and CR3022, respectively, at 100% neutralization [92
]. These results suggest a potential use of CR3014 in combination with CR3022 for prophylaxis of SARS CoV infections in humans. However, one should note that currently there is no available animal model of the SARS CoV infection that results in death as in humans.
Two other hmAbs (201 and 68) were derived from transgenic mice with human Ig genes and evaluated in a murine model of SARS CoV infection [93
]. One of these antibodies 201 bound within the RBD of the S protein at amino acid residues 490–510 while the other one 68 bound to a region containing residues 130–150. In a microneutralization assay based on protection to cytopathic effects the IC50
for 201 was about 0.2 µg/ml [93
]. Mice that received 40 mg/kg of these antibodies prior to challenge with the SARS CoV were completely protected from virus replication in the lungs, and doses as low as 1.6 mg/kg offered significant protection. These antibodies have potential as therapeutics and research tools [93
Recently, a novel cross-reactive potent SARS CoV-neutralizing hmAb, m396, was identified by using a fragment containing residues 317 through 518 as a selecting antigen for panning of a large human antibody library constructed from the B lymphocytes of healthy volunteers [94
]; this fragment was previously identified to contain the RBD [95
], which is a major SARS CoV neutralization determinant [84
]. Because the SARS CoV caused two outbreaks - the epidemic in late 2002/early 2003 and a second outbreak in the winter of 2003–2004 by an independent animal-to-human transmission – the antibody m396 was tested against isolates from both outbreaks and from animal hosts that initiated these outbreaks. The GD03 strain, which was isolated from an index patient of the second outbreak, was reported to resist neutralization by the hmAbs 80R and S3.1 which can potently neutralize isolates from the first outbreak. It was found that m396 and another antibody, S230.15, potently neutralized GD03 and representative isolates from the first SARS outbreak (Urbani, Tor2) and from palm civets (SZ3, SZ16) [104
]. These antibodies also protected mice challenged with the Urbani, or recombinant viruses bearing the GD03 and SZ16 S glycoproteins. Both antibodies competed with the SARS CoV receptor, ACE2, for binding to the RBD suggesting a mechanism of neutralization that involves interference with the SARS CoV-ACE2 interaction. These antibodies are the first identified hmAbs, which exhibit cross-reactivity against isolates from the two SARS outbreaks and palm civets, and could have potential applications for diagnosis, prophylaxis and treatment of SARS CoV infections.
NAbs directed to S inhibit SARS CoV entry either by interfering typically with the S RBD-receptor interactions [87
] or by other mechanisms including binding to other portions of S. A human scFv, B1, was identified, which recognizes an epitope on S2 protein located within amino acids 1023–1189 [105
]. This antibody recognized SARS pseudovirus in vivo and competed with SARS sera for binding to SARS CoV with an equilibrium dissociation constant, Kd
= 105 nM. The B1 also had potent neutralizing activities against infection by pseudovirus expressing SARS CoV S protein in vitro. Other mechanisms of SARS CoV infection inhibition could include steric hindrance that indirectly prevents virus attachment to receptors and binding to entry intermediates. Mechanisms that could operate in vivo, and for lack of data will not be discussed here, are related to the antibody biological effector functions conferred by the antibody Fc, e.g., antibody-dependent cellular cytotoxicity (ADCC).
The S-ACE2 interactions can be blocked by antibodies targeting either S or ACE2. Indeed, it was found that antibodies to ACE2 but not an anti-ACE1 antibody blocked viral replication on Vero E6 cells [106
]. Because the receptor is a host molecule, which does not mutate, the use of antibodies targeting receptor molecules may prevent the generation of resistant mutants. However, it appears that for SARS CoV infection, which is an acute infection, generation of resistant mutants may not be a significant problem although the recent identification of bats as a natural reservoir of SARS CoV suggests the possibility for large variations in sequences of viruses that could be transmitted to humans [107
]. In addition, such anti-ACE2 antibodies could affect the function of ACE2-expressing cells. Experiments in animal models are required to find whether SARS CoV infection in presence of nhmAbs will lead to generation of neutralization escape mutants and whether anti-ACE2 nhmAbs antibodies have deleterious effects on the host.