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protein

You Don’t Need the Whole Antibody

1 October 2020 by Gertrud U. Rey

by Gertrud U. Rey

Antibodies are large proteins that are made by B cells of the adaptive immune system. Most people think that antibodies function only as a whole molecule, but some of the individual fragments of an antibody can also bind and neutralize antigens. 

An antibody consists of two heavy chains and two light chains that assemble into a Y-shaped structure (left side of Figure). The stem of the Y is known as the “fragment crystallizable” (Fc) portion and is composed of two heavy chains. The two arms of the Y are known as the “fragment antigen-binding” (Fab) portions and are each composed of one heavy chain and one light chain. As its name suggests, the top half of each Fab fragment is the antigen-binding region of the antibody, and it is variable – meaning that it varies between antibodies that are produced by different B cells. The bottom half of each Fab fragment and the entire Fc region are constant, meaning that they are identical in all antibodies of the same isotype, but differ in antibodies of different isotypes. For example, the constant regions are identical in all IgG antibodies but differ between IgG and IgA antibodies. 

In an effort to identify anti-SARS-CoV-2 antibodies suitable for preventing and treating SARS-CoV-2 infection, the authors of a recent publication screened 100 billion different anti-SARS-CoV-2 antibody candidates for their ability to bind and/or neutralize SARS-CoV-2. This eventually led to the discovery of “ab8,” an antibody fragment consisting of a variable heavy (VH) region and having particularly potent SARS-CoV-2 binding specificity and neutralization activity. To increase the binding avidity of ab8 (i.e., the stability of its interaction with an antigen) and extend its longevity in the human body, the authors fused this fragment to the Fc domain of human IgG1, an abundant and stable type of human antibody. This produced the molecule hereinafter referred to as “VH-Fc ab8″ (right side of Figure).  

The authors found that VH-Fc ab8 can bind various conformations of the SARS-CoV-2 spike protein, including when the spike protein is bound to a cell surface. VH-Fc ab8 can also bind to and neutralize six different SARS-CoV-2 isolates having different amino acid changes in the receptor-binding domain, suggesting that it is broadly cross-reactive. Notably, it does not bind to human cells, meaning that it does not seem to interfere with normal cellular functions.

As a next step, the authors evaluated the ability of VH-Fc ab8 to prevent SARS-CoV-2 infection in mice. If given to mice before they were infected with SARS-CoV-2, VH-Fc ab8 inhibited viral replication at all doses tested, but it only neutralized virus at the highest dose of 36 mg/kg. Although these results were encouraging, it is often difficult to interpret data obtained in mice in terms of clinical relevance in humans, because mice don’t develop the COVID-19-related disease pathologies observed in humans. Hamsters more closely imitate human SARS-CoV-2 infection in the lung, suggesting that they could be a useful mammalian model for COVID-19. VH-Fc ab8 caused significantly reduced levels of infectious virus in the lung, nasal mucosa, and saliva of hamsters when administered one day before (i.e., “prophylactically”) or six hours after SARS-CoV-2 infection (i.e., “therapeutically”) compared to untreated control animals, suggesting that it could be used to both prevent and treat SARS-CoV-2 infection. Although VH-Fc ab8 led to greater reduction of virus levels when given prophylactically than when given therapeutically, therapeutic administration still led to significantly decreased viral loads in treated animals compared to untreated control animals, even at very low doses. VH-Fc ab8 not only alleviated pneumonia and reduced lung viral loads in hamsters, but it also reduced virus shedding in the upper airway, which could help with reducing transmission. 

The authors also found that when they gave hamsters the same dose of either VH-Fc ab8 or IgG1 ab1 – a full-sized version of the antibody, and then examined their concentrations in the serum five days later, levels of VH-Fc ab8 were significantly higher than those of the full-sized antibody. This suggests that the systemic distribution of VH-Fc ab8 is more long-lived than that of a full-sized antibody. 

Although small animal models can provide key insights into the pathogenic mechanisms of viral infections, they are often poor predictors of human disease outcomes. The therapeutic timeline followed in the hamster experiments (i.e., administration of VH-Fc ab8 six hours after infection) would also be difficult to reproduce in humans because therapeutic drugs are not usually administered until well after symptom onset. Therefore, it would be difficult to determine whether the therapeutic effect of VH-Fc ab8 observed in hamsters would be the same in humans. 

That being said, there are clear advantages to using antibody fragments instead of whole antibodies. Their small size allows them to penetrate more efficiently to sites of infection and bind antigens more easily and with more specificity. Smaller molecules also diffuse more easily through tissues, meaning that they could be administered by routes other than injection, such as by inhalation. Furthermore, because the molecular weight of VH-Fc ab8 is only about half that of a full-sized antibody, smaller quantities would be needed to obtain the same number of molecules, meaning that antibody fragment therapeutics could be more easily mass-produced. 

There is no question that we are in dire need of an effective therapeutic drug to treat SARS-CoV-2 infection. If the results observed in these animal experiments can be duplicated in humans, VH-Fc ab8 would be an attractive option for both treating and preventing SARS-CoV-2 infection. 

Filed Under: Gertrud Rey, Uncategorized Tagged With: animal model, antibody, Fab fragment, Fc fragment, fragment, heavy chain, molecule, neutralizing, prophylactic, protein, SARS-CoV-2, therapeutic, treatment

TWiV 394: Cards in a hand

19 June 2016 by Vincent Racaniello

Vincent and Alan speak with Erica Ollmann Saphire about her career and her work on understanding the functions of proteins of Ebolaviruses, Marburg virus, and other hemorrhagic fever viruses, at ASM Microbe 2016 in Boston, MA.

You can find TWiV #394 at microbe.tv/twiv, or listen or watch the video below.

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Filed Under: This Week in Virology Tagged With: cryoEM, ebolavirus, filovirus, marburg virus, protein, protein structure, structural biology, viral, virology, virus, viruses, X-ray structure

TWiV 220: Flu watches the clock while T7 gets a CAT scan

17 February 2013 by Vincent Racaniello

On episode #220 of the science show This Week in Virology, Vincent, Rich, Alan, and Kathy discuss regulation of influenza virus replication by splicing, and the bacteriophage T7 random walk.

You can find TWiV #220 at www.microbe.tv/twiv.

Filed Under: This Week in Virology Tagged With: bacteriophage t7, cryo-electron tomography, Cryo-ET, influenza, NEP, NS1, NS2, nuclear export, protein, rna splicing, viral, virology, virus

TWiM 30: Unraveling melioidosis and insulin resistance

4 April 2012 by Vincent Racaniello

On episode #30 of the science show This Week in Microbiology, Vincent, Elio, and Michael review how a toxin from Burkholderia pseudomallei inhibits protein synthesis, and the role of the gut microbiome in modulating insulin resistance in mice lacking an innate immune sensor.

You can find TWiM #30 at microbeworld.org/twim.

Filed Under: This Week in Microbiology Tagged With: burkholderia, gut, immune, innate, insulin, melioidosis, mice, microbiome, protein, pseudomallei, resistance, sensor, synthesis, toxin

Viral bioinformatics: Sequence searcher

4 April 2011 by Vincent Racaniello

virology toolboxThis week’s addition to the virology toolbox was written by Chris Upton

Sequence Searcher is a Java program that allows users to search for specific sequence motifs in protein or DNA sequences. For example, it can be used to identify restriction enzyme cleavage sites or find similar sequence patterns among multiple sequences. Most searches run in a few seconds.

Sequence Searcher is part of the Virology.ca suite of programs available at the University of Victoria.

Help files:

  • Quick start
  • How-to

Some of the key features of Sequence Searcher include:

  • Searching through multiple sequences
  • Use of regular expressions or fuzzy search patterns.
  • Searching for patterns on both strands of a DNA sequence
  • Graphical representation of results and ability to save search results
  • It can run on multiple computer platforms (Java)

For DNA, the searches are conducted by finding the motif within a sequence from the 5’ to 3’ end on the top strand. The searches are also processed from the 5’ to 3’ end of the bottom strand. As a result, bases are numbered from 1 starting at the 5’ at either the top or bottom strand.

Regular expression and fuzzy pattern searches are available:

Fuzzy searches provide the option for the program to allow a certain number of mismatches from a sequence input at any position.  Note that the maximum number of mismatches that the program allows is 40% of the length of the sequence motif.

Regular expression allows for inputs of precise motifs along with considerable user-specified flexibility at specific positions.

figure 1

Figure 1. The input tab is where you can import DNA or protein sequences (must be in FASTA format) and type in the specific pattern to search within in the sequence(s). The search type can be selected as “Regular expression” or “Fuzzy” by using the drop down menu.

figure 2

Figure 2. When a search has been completed, the results tab is presented in a table format. The results in the table can be sorted depending on the column header (sequence, match, start, stop, confidence, and strand). The results can also be filtered by sequence and strand by selecting the drop down menus at the top.

Marass, F., & Upton, C. (2009). Sequence Searcher: A Java tool to perform regular expression and fuzzy searches of multiple DNA and protein sequences BMC Research Notes, 2 (1) DOI: 10.1186/1756-0500-2-14

Filed Under: Toolbox Tagged With: bioinformatics, DNA, genomics, java, nucleotide sequence, protein, sequence motif, sequence searcher, viral, virology, virus

Viral bioinformatics: Introduction to multiple sequence alignment

15 October 2010 by Vincent Racaniello

This week’s addition to the virology toolbox was written by Chris Upton

Generating multiple sequence alignments (MSA) is one of the most commonly used bioinformatics techniques. The “sequences” to be compared can be DNA (promoters, genes, genomes) or proteins. Note that the length and number of sequences to be aligned has an impact on the methods (algorithms) that can be used; what is suitable for aligning 20 proteins probably won’t work for alignment of 5 poxvirus genomes (200 kb each).

Some useful links:

  • Wikipedia: multiple sequence alignment
  • Wikipedia: sequence alignment
  • Wikipedia: list of sequence alignment software
  • Protein Multiple Sequence Alignment: Book chapter by Chuong B. Do and Kazutaka Katoh
  • Sequence alignment: Lecture notes by Per Kraulis
  • Another list of tools

So you see, there lots of options (did you say: “too many!”?). Further confusion may arise because 1) the same algorithm may be used in many different software programs, and 2) referencing a software package may give no clue to the algorithm used. For many molecular biologists, Clustal is synonymous with sequence alignment. However, newer algorithms such as T-Coffee and MUSCLE are often offered in current software packages, and may be faster and more accurate.

Specialized alignment tools are almost always needed for long, genome sized DNA sequences.

In this set of posts, I’ll provide some information on favorite general MSA tools (that are free) that should be useful to the average molecular virologist. The lists noted above provide a multitude of tools, but many are for specific analyses.

Filed Under: Toolbox Tagged With: bioinformatics, clustal, multiple sequence alignment, muscle, nucleic acid, protein, t-coffee, Toolbox, viral, virology, virus

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by Vincent Racaniello

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